1
Biophysics and p o ein co ona analysis o Janus cyclodex in-DNA
nanocomplexes. E icien cellula ans ec ion on cance cells
M. Ma ínez-Neg oa, G. Ca acciolob, S. Palche ib, D. Pozzib, A. L. Cap io ic, C.
Ca alie ec, A. Laganàc, C. O iz Melle d, J. M. Beni oe, J. M. Ga cía Fe nándeze, E.
Aica a, E. Junque aa,*
aG upo de Química Coloidal y Sup amolecula , Depa amen o de Química Física I,
Facul ad de Ciencias Químicas, Uni e sidad Complu ense de Mad id, 28040-Mad id,
Spain
bDepa men o Molecula Medicine, “Sapienza” Uni e si y o Rome, Viale Regina
Elena 291, 00161-Rome, I aly
cDepa men o Chemis y, “La Sapienza” Uni e si y o Rome, Pzle Aldo Mo o 5,
00185-Rome, I aly
dDepa amen o de Química O gánica, Facul ad de Química, Uni e sidad de Se illa, c/
P o eso Ga cía González 1, 41012-Se illa, Spain
eIns i u o de In es igaciones Químicas (IIQ), CSIC – Uni e sidad de Se illa, A da.
Amé ico Vespucio 49, 41092-Se illa, Spain
Co esponding Au ho
*Fax: +34913944135. E-mail: [email p o ec ed]
2
ABSTRACT
The sel -assembling p ocesses unde lining he capabili ies o acially di e en ia ed
(“Janus”) polyca ionic amphiphilic cyclodex ins (paCDs) as non- i al gene
nanoca ie s ha e been in es iga ed by a plu idisciplina y app oach. Th ee
ep esen a i e Janus paCDs bea ing a common e adecahexanoyl mul i ail domain a
he seconda y ace and di e ing in he opology o he clus e o amino g oups a he
p ima y side we e selec ed o his s udy. All o hem compac pEGFP-C3 plasmid
DNA and p omo e ans ec ion in HeLa and MCF-7 cells, bo h in absence and in
p esence o human se um. The elec ochemical and s uc u al cha ac e is ics o he
paCD-pDNA complexes (CDplexes) ha e been s udied by using ze a po en ial, DLS,
SAXS, and c yo-TEM. paCDs and pDNA, when assembled in CDplexes, ende
e ec i e cha ges ha a e lowe han he nominal ones. The CDplexes show a sel -
assembling pa e n co esponding o mul ilamella lyo opic liquid c ys al phases,
cha ac e ized by a lamella s acking o bilaye s o he CD-based ec o s wi h anionic
pDNA sandwiched among hem. When exposed o human se um, ei he in he absence
o in he p esence o pDNA, he su ace o he ca ionic CD-based ec o becomes
coa ed by a p o ein co ona (PC) whose composi ion has been analysed by nanoLC-
MS/MS. Some o he CDplexes he ein s udied showed mode a e- o-high ans ec ion
le els in HeLa and MCF-7 cance cells combined wi h mode a e- o-high cell iabili ies,
as de e mined by FACS and MTT educ ion assays. The ensemble o da a p o ides a
de ail pic u e o he paCD-pDNA-PC associa ion p ocesses and a a ional base o
exploi he p o ein co ona o a ge ed gene deli e y on u u e in i o applica ions.
KEYWORDS: CDplexes, e ec i e cha ge a io, mul ilamella phases, cellula
ans ec ion, cy o oxici y, p o ein co ona
3
1. In oduc ion
Nucleic acids (DNA o RNA) ha e become in he las decade an a ac i e sou ce o
he apeu ic agen s [1]. The in e play o a di ec s uc u e-ac i i y ela ionship and a
highly speci ic mode o ac ion heo e ically pe mi exploi ing he cellula machine y in a
p edic able ashion o ei he s imula e o silence he exp ession o i ually any p o ein,
wi h educed oxici y and ewe side e ec s as compa ed wi h classical d ugs. This is
he basis o gene he apy (GT), an exci ing b anch o medicine ha ies o cu e diseases
(gene ic, neu ologic, ca dio ascula , in ec ious, o ca cinogenic, e c.) a a molecula
le el, by epai ing damaged cellula DNA, ei he by in oducing (by means o a plasmid
DNA) and exp essing a copy o he a ec ed o missing gene in o he cells [2-8] o by
inse ing a small in e e ing RNA (siRNA) o silence and s op he exp ession o an
abno mal gene, esponsible o he cell diso de [9-12]. Bo h app oaches, ei he ha one
based on plasmid DNAs o on siRNAs, cons i u e nowadays he main s a egies o GT
on seeking o e ec i ely es o e heal hy cell unc ioning.
Con en ional d ugs consis o bioac i e species and a ca ie , he o me being
no mally he key o he design s a egies. Howe e , in he case o biomolecula d ugs,
such as nucleic acids, he ca ie becomes a decisi e p o agonis . In he ee s a e,
nucleic acids a e easily deg aded by nucleases in biological luids [13,14], and hei
memb ane-c ossing abili ies and cellula up ake a e se iously limi ed by hei nega i e
cha ge, inhe en ly la ge size, and igidi y [15]. Fo mula ion wi h app op ia e deli e y
sys ems is hus essen ial o nucleic acids o o e come he physiological ba ie s, each
he a ge in a ully unc ional o m and ca y ou he designed he apeu ic unc ion.
Vi al gene ec o s [16,17] we e i s de eloped owa ds his end due o hei high
e ec i eness. Ye , he use o i al ec o s bea inhe en isks, including immunogenici y
and oncogenici y, which has boos ed esea ch on he design, syn hesis, and
4
cha ac e iza ion o non- i al ca ie s ha may combine high ans ec ion e iciencies
wi h low cy o oxici y le els [18,19]. In addi ion, o in i o applica ions, hese ec o s
mus emain s able in he p esence o blood o se um and, ideally, should be amenable
o su ace deco a ion wi h selec i e g oups in o de o ecognize he a ge cells. The
majo i y o non- i al ec o o mula ions on eco d a e based on ca ionic lipids [19,20],
polyme s [21], nanopa icles [22], and polysaccha ides [12,23]. Mo e ecen ly, gene
deli e y sys ems based on 3D molecula amewo ks wi h p ecisely de ined chemical
s uc u es, sizes, shape symme y and unc ional g oup dis ibu ions ha e been
de eloped [24-26]. Thei in insic monodispe se cha ac e allows conduc ing sys ema ic
s udies on he in luence o ec o modi ica ions on he sup amolecula in e ac ions wi h
nucleic acids and he consequences in ans ec ion e iciencies [1].
Wi hin he con ex o molecula gene ec o s, acially di e en ia ed (“Janus”)
mac ocyclic en i ies [24,26,27], among which cyclodex in (CD)-based de i a i es a e
pa adigma ic examples, ha e conque ed a p ominen posi ion [28,29]. CDs a e
biocompa ible cyclic oligosaccha ides consis ing o α-1,4-linked glucopy anose uni s
ha de ine a cone-shape opology wi h well-di e en ia ed aces. The Janus ea u e can
be impa ed by bidi ec ional unc ionaliza ion wi h polyca ionic g oups on one im and
hyd ophobic chains on he opposi e im. Mul ihead-mul i ail polyca ionic amphiphilic
CDs (paCDs) wi h sel -assembling p ope ies and biomime ic cell-memb ane-c ossing
ap i udes, esembling bo h ca ionic lipids and ca ionic polyme s, a e hus accessed
[30,31]. In he p esence o nucleic acids, hey spon aneously o m well-de ined
sup amolecula nanocomplexes (he eina e e e ed o as CDplexes) whe e he gene
ma e ial is p o ec ed om deg ada ion by enzyma ic agen s. Analogously o he well-
known lipoplexes and polyplexes [32], CDplexes can elici cellula up ake in
physiological media and p omo e ans ec ion [30,31,33,34].
5
Whe eas in i o ans ec ion o immo alized cul u ed cells can be gene ally
conduc ed in se um- ee medium, some ex- i o and any in i o GT p o ocol will imply
exposing o he nucleic acid-en apping nanosys ems o o he biomolecules p esen in
biological luids. Non-speci ic in e ac ions wi h hese biomolecules may hen decisi ely
a ec hei a e in a biological en i onmen [35,36]. Indeed, once adminis e ed in i o,
he nano ec o s a e exposed o he biological luids om which hey adso b p o eins
and o he componen s, losing he ba e ec o iden i y o o m a biologically ac i e
co ona, known as he p o ein co ona (PC) [37,38]. This PC plays an impo an ole in
bo h he cellula ecogni ion [39] and cell-memb ane c ossing capabili ies
(in e naliza ion p ocess) o he nano ec o [40]. I has also a clea e ec on i s ime o
ci cula ion in he blood, so ha knowing he p o eomic p o ile o he co ona
su ounding a gene ec o is c ucial o op imize he ans ec ion p ocess. Fo ins ance,
ce ain p o eins like opsonins and, specially, immunoglobulins, ib inogen and
complemen p o eins a e ecognized by he mac ophages a o ing he phagocy osis and
he apid clea ance o he ec o om he bloods eam. On he o he hand, he
adso p ion o apolipop o eins and se um albumin (dyopsonins) p omo e p olonged
blood ci cula ion hal -li e [41]. Success ul deli e y o he apeu ic genes in o cells will
he e o e equi e no only a high con ol o he physical-chemical pa ame e s o he
ec o and ec o -DNA nanocomplex, such as e ec i e cha ges, chemical composi ion,
size and s uc u e, bu also a deep knowledge on hei in e ac ion wi h se um p o eins,
he composi ion o he esul ing PC and i s impac in ans ec ion capabili ies and
oxici y. No wi hs anding, up o da e, mos s udies in his ield a e cen e ed on he PC
ha o ms a ound ino ganic and o ganic nanopa icles [40,42-44], wi h only a ew
in es iga ions e alua ing he PC adso bed on ec o -DNA complexes [45,46].

6
This wo k is speci ically ocused on he biophysical and biochemical
cha ac e iza ion o Janus paCD-pDNA CDplexes and hei p o ein co ona inge p in s
as a unc ion o he molecula paCD ec o opology. We ha e chosen h ee di e en
paCD ep esen a i es wi h a -cyclodex in (CD) co e, namely compounds ADM70,
ADM105 and PBO234 (Scheme 1). The h ee molecula ec o s sha e a C7-
symme ical ski - ype Janus a chi ec u e wi h a mul i ail lipophilic domain o med by
ou een hexanoyl g oups ha es e i y he seconda y hyd oxyls, di e ing in he
a chi ec u e o he mul i alen ca ionic clus e ins alled a he p ima y posi ions. Thus,
ADM70 displays a dend i ic p esen a ion o ou een p ima y amino g oups, ADM105
a linea a angemen o se en seconda y and se en p ima y amino g oups and PBO234
a hep a alen clus e o p ima y amino g oups. ADM70 and ADM105 addi ionally
inco po a e a bel o se en hiou ea g oups a he p ima y ace b anches. All hese
s uc u al elemen s ha e been p e iously ound o impa good DNA compac ion
abili ies o molecula gene ec o s as well as good ans ec ion capabili ies o he
co esponding ec o -DNA complexes in cellulo and in i o [33,34]. We ha e i s
conduc ed a deep elec ochemical and s uc u al cha ac e iza ion o he ba e nano ec o
and pDNA-loaded Janus paCD assemblies by using high p ecision expe imen al
echniques including ze a po en ial, aga ose gel elec opho esis, dynamic ligh sca e ing
(DLS), small angle X- ay sca e ing (SAXS) and c yo- ansmission elec on mic oscopy
(c yo-TEM). Gi en ha wha cells “see” and p ocess in in i o si ua ion is no he ba e
complexes bu he CDplexes-PC bioen i y o med, we ha e e alua ed he PC de eloped
a he su ace o CDplexes o mula ed wi h he h ee paCDs he ein used when hey
in e ac wi h human plasma (HP), by means o nano liquid ch oma og aphy andem
mass spec ome y (nanoLC-MS/MS). Finally, ans ec ion pe o mances o he
CDplexes and le els o cellula oxici y we e u he in es iga ed in i o by
7
luo escence-ac i a ed cell so ing (FACS) and educ ion o 3-(4,5-dyme hyl hiazol-2-
y1)-2,5-diphenyl e azolium b omide (MTT assay), espec i ely. The inal goal o his
plu idisciplina y app oach is ob aining a global pic u e o he op imal equi emen s o
he p epa a ion o he CDplexes, in an a emp o opening al e na i e ails ha p o ide
non- i al ec o s wi h imp o ed ou pu s o bioa ailabili y and ans ec ion e iciency
(TE).
Scheme 1. S uc u al cha ac e is ics o ADM70, ADM105 and PBO234.
2. Expe imen al sec ion
2.1 Ma e ials
The Janus paCDs used in his s udy, ADM70, ADM105 and PBO234 (Scheme 1),
we e p epa ed ollowing he p ocedu es p e iously epo ed [47]. B ie ly, he syn hesis
o compound PBO234 was accomplished in ou s eps (i-i ) om comme cially
a ailable -cyclodex in by (i) hep ab omina ion a he p ima y hyd oxyl im wi h he
N-b omosuccinimide (NBS)/ iphenylphosphine (TPP) sys em [48], (ii) cesium
ca bona e p omo ed nucleophilic displacemen o b omine by N-Boc-cys eamine [49],
=
== =
=
+
ADM70
ADM105
PBO234
ADM70 ADM105 PBO234
+
CD paCD
8
(iii) acyla ion o he ou een seconda y hyd oxyls by eac ion wi h hexanoic anhyd ide
in N,N-dime hyl o mamide (DMF) and N,N-dime hylaminopy idine (DMAP) as a non-
nucleophilic base ca alys and (i ) inal acid-p omo ed hyd olysis o he e -bu yl
ca bama e g oups. Compounds ADM105 and ADM70 we e ob ained om PBO234
a e iso hiocyana ion o he se en amino g oups wi h hiophosgene [47] ollowed by
hiou ea coupling eac ions wi h mono-N-Boc-e hylenediamine o N,N”-di-Boc-
e hylene iamine, espec i ely, and subsequen Boc emo al. The physicochemical da a
o he h ee polyca ionic amphiphilic cyclodex ins we e consis en wi h hose
p e iously epo ed [47]. Mos signi ican ly, he 1H and 13C NMR spec a showed he
ypical single-spin sys em o ully C7-symme ical molecules.
pEGFP-C3 Plasmid DNA (pDNA) was ex ac ed om compe en E. Coli bac e ia
p e iously ans o med wi h pEGFP-C3, he ex ac ion being ca ied ou using
GenElu e HP Selec plasmid Gigap ep Ki (Sigma Ald ich) ollowing a p o ocol
p e iously desc ibed [50,51]. Sodium sal o cal hymus DNA (c DNA), p o ided by
Sigma-Ald ich, was used as linea DNA o de e mine he e ec i e cha ge ( ) o he
ca ionic ec o .
Human plasma (HP) whole blood p o ided by he Depa men o Expe imen al
Medicine o La Sapienza Uni e si y o Rome, was ob ained by enipunc u e o en
heal hy olun ee s. Mixed plasma was aliquo ed and s o ed a -80 ºC in p o ein LoBind
ubes un il u he use. Fo analysis, he aliquo s we e hawed a 4 ºC and hen allowed
o wa m a oom empe a u e. Human ce ical cance cell line (HeLa), de i ed om
human ce ix adenoca cinoma, and human b eas cance cell line (MCF-7), de i ed
om human pleu al e usion b eas cance me as asis, we e pu chase om ATCC
(Manassas, VA, USA). HeLa cells we e main ained in Eagle’s Minimum Essen ial
Medium (EMEM) supplemen ed wi h 2 mM L-glu amine, 100 IU/mL
+
CD
q
9
penicillin/s ep omycin, 1 mM sodium py u a e, 10 mM HEPES, 1.5 mg/L sodium
bica bona e, and 10% e al bo ine se um (FBS). MCF-7 cells we e main ained in
Eagle’s Minimum Essen ial Medium (EMEM) supplemen ed wi h 0.01 mg/ml human
ecombinan insulin and 10% e al bo ine se um (FBS).
2.2 Me hods
2.2.1 P epa a ion o CDplexes. paCD-DNA complexes we e o med by mixing he
co ec amoun s o aqueous solu ions o he paCDs and o DNAs (ei he c DNA o
pDNA) in HEPES 20 mM (pH = 7.4). The inal solu ions we e le du ing 20 min p io
o ca y on he expe imen s. pDNA concen a ions we e op imized o i he op imum
condi ions o each expe imen al echnique. CDplex composi ion can be exp essed
ei he in e ms o he masses a io
 
CD DNA
m / m

, be ween mass o he gene ec o (
) o plasmid DNA
 
DNA
m
, o he e ec i e cha ge a io (ρe ), be ween he paCD
and pDNA e ec i e cha ges.
2.2.2. Incuba ion o paCDs wi h HP, in absence and p esence o pDNA. paCDs
we e mixed wi h HP (1:1 / ) and we e incuba ed a 37 ºC o 1 h. A e incuba ion, he
samples we e cen i uged h ee imes du ing 15 min a 14000 pm in o de o wash he
sample and emo e all he molecules no bound o he complex. The same p ocedu e
was ollowed wi h he CDplexes.
2.2.3. Me hods o he cha ac e iza ion o paCD/pDNA CDplexes. Uncomplexed
plasmid DNA along wi h he paCD/pDNA CDplexes (a se e al
 
CD pDNA
m / m
a ios)
we e loaded on o 1% aga ose gel and un o 30 min a 100 mV in 1x TAE (T is-HCl,
Ace a e and EDTA) bu e . In he Aga ose gel elec opho esis expe imen s, ully
paCD/pDNA complexes appea ed as luo escen band in he wells o he gel, while
CD
m
16
cha ged complex, as equi ed o c oss he nega i e cell memb ane in an e icien cell
ans ec ion p ocess. F om Eq. (2), i is easily deduced ha = 1 equi es:
(3)
Thus, i he isoneu ali y a io
 
CD linea DNA
m / m

is expe imen ally de e mined o a
complex o med by a Janus CD-based ec o and a comme cial linea DNA ( o which
= -2/bp), he e ec i e posi i e cha ge o he ec o ( ) can be ob ained.
Once his cha ge is known, he nega i e e ec i e cha ge o he plasmid ( ) can
be s aigh o wa dly de e mined om he expe imen al alue o
 
CD pDNA
m / m

o a
complex o med by he same CD based ec o bu wi h a plasmid DNA ins ead o he
linea DNA, on he same expe imen al condi ions, as ollows:
(4)
Elec ochemical echniques a e, among o he s, he mos adequa e ools o de e mine
his elec oneu ali y a io, aga ose gel elec opho esis, and, mo e p ecisely, ze a
po en ial, being he mos ecommended. Aga ose gel elec opho esis in o ms abou he
compac ion le el o pDNA by he ec o s. Fig.1 epo s hese expe imen s a h ee
di e en
 
CD DNA
m / m
a ios (whi e numbe s on lanes 2-4), o he h ee paCDs used in
his wo k (ADM70, ADM105 and PBO234). Uncomplexed pDNA (lane 1) was used
as a posi i e con ol. Resul s epo ed in Fig. 1 e eal ha pDNA is e icien ly
compac ed by he paCD molecula ec o s, since he luo escen bands disappea ac oss
he gel lanes as long as
 
CD DNA
m / m
a io inc eases. As can be in e ed om Fig. 1, he
e

1
, ,linea CD CD
e CD e DNA bp
linea DNA
mM
qq mM


 


,linea e DNA
q
,e CD
q
,pe DNA
q
,p ,
bp
CD
e DNA e CD
pDNA CD
mM
qq
mM







17
isoneu ali y was each a
 
CD DNA
m / m
a ios below 0.4, 0.7 and 0.5 o ADM70-
pDNA, ADM105-pDNA and PBO234-pDNA CDplexes, espec i ely.
Wi h he aim o de e mining wi h highe p ecision he elec oneu ali y a io, ze a
po en ial was measu ed as a unc ion o
 
CD DNA
m / m
(Fig. 2), co e ing he ange
wi hin which pDNA is e ec i ely compac ed by each paCD, acco ding o he esul s
shown in Fig. 1. The elec oneu ali y a io
 
CD DNA
m / m

can be de e mined as he
 
CD DNA
m / m
alue whe e a sign in e sion on he cha ge occu s in he

-po en ial
sigmoidal p o iles.
Fig. 1. Gel elec opho esis esul s o (a) ADM70-pDNA CDplexes, (b)
ADM105-pDNA CDplexes, and (c) PBO234
-pDNA CDplexes. Lane 1:
uncomplexed pDNA (posi i e con ol). Lanes 2-4: CDplexes a di e en
mCD/mDNA mass a ios (whi e numbe s a he bo om o he lanes).
pDNA mCD/mDNA
0.4
0.7
1.6
0.7
1.6
3.0
0.5
1.0
2.0
b)
c)
a)
18
Fig. 2. Plo o ζ po en ial s mCD/mDNA CDplex composi ion, o di e en samples wi h
c DNA (a) and pDNA (b). Black, ed and blue symbols co espond o ADM70-pDNA,
ADM105-pDNA and PBO234-pDNA CDplexes, espec i ely.
Table 1 epo s he esul s ob ained o CDplexes o mula ed wi h ADM70,
ADM105 o PBO234 and ei he c DNA o pDNA. Wi h hese
 
CD DNA
m / m

alues
and ollowing he p ocedu e abo e explained, he e ec i e cha ges o bo h he Janus
CD based ca ionic ec o s and he pDNA he ein used, and , we e
calcula ed and collec ed in Table 2. The di e ences encoun e ed be ween nominal and
e ec i e cha ges in bo h he ca ionic ec o s and he anionic pDNA a e ema kable.
Thus, he ne posi i e cha ges a ailable o in e ac ion wi h pDNA a e a ound 33% o
he nominal one, assuming ull p o ona ion o he amino g oups, o he e adecaamine
de i a i es ADM70 and ADM105, and only 25% o hep amine PBO234. This
scena io is simila o ha ound o some CD poly o axane-based ec o s [12] bu
sha ply di e en om ha encoun e ed o ca ionic lipid gene ec o s bea ing
qua e na y amino g oups, which no mally yield hei o al nominal posi i e cha ges
wi hin a ange o 10 % o unce ain y [19,51,57]. On he o he hand, he plasmid used
he ein seems o be qui e supe coiled a he expe imen al condi ions used, since i s
a ailable nega i e cha ge pe bp is a away om he nominal alue (-2/bp), yielding
,e CD
q
,p

e DNA
q
0,0 0,2 0,4 0,6 0,8
-20
0
20
 (mV)
mCD/mDNA
b)
0 3 6 9
-30
0
30
 (mV)
mCD/mDNA
a)
19
a ound 7%, 13% and 5% o i s nominal cha ge when being compac ed by ADM70,
ADM105 and PBO234, espec i ely. This beha iou , o en ound in lipoplexes,
con i ms ha plasmids usually e ain an impo an pe cen age o ca ionic sodium
coun e -ions (Na+). This is a p io i a a ou able ea u e o he use o hese mac ocyclic
ec o s as sa e and e ec i e ehicles o nucleic acids, since he weake he anionic
cha ac e o he DNA, he lowe he amoun o ca ionic ec o needed o o mula e he
nanocomplexes, hus dec easing he isk o cy o oxici y o he ec o . F om he paCD
and pDNA e ec i e cha ges hus ob ained, e ec i e cha ge a ios (

e ) a ound 5- old
( o ADM70 and PBO234) o 2- o 3- old ( o ADM105) o he nominal ones we e
calcula ed using Eq. (2).
Table 1. Values o isoneu ali y a ios
 
CD DNA
m / m

o he CDplexes o med by
ADM70, ADM105 and PBO234 wi h ei he c DNA o pDNA.
ADM70-DNA
ADM105-DNA
PBO234-DNA
c DNA
3.0
2.9
5.7
pDNA
0.19
0.38
0.25
Values es ima ed wi h a 5% e o
Table 2. Nominal and e ec i e cha ges o paCDs and pDNA.
ADM70
ADM105
PBO234
nom,CD
q
14
14
7
e ,CD
q
4.6
4.7
1.7
e ,CD nom,CD
q / q

0.33
0.33
0.25
nom,pDNA
q / bp

-2
-2
-2
e ,pDNA
q / bp

-0.13
-0.26
-0.09
e ,pDNA nom,pDNA
q / q

0.07
0.13
0.05

e /

nom
5.1
2.6
5.4
Values es ima ed wi h an 8% e o
Ha ing in o ma ion o he s uc u e o he complex is impo an o ind he bes non-
i al ec o s and o p omo e i s use in in i o gene he apy. The s uc u e o he paCD-
pDNA CDplexes in concen a ed samples was in es iga ed by SAXS a se e al
20
e ec i e cha ge a ios o which CDplexes a e po en ially ac i e as gene ans ec ion
ec o s (

e > 1). C yo-TEM was also used as a suppo ing echnique. Fig. 3 shows he
co esponding SAXS di ac og ams (In ensi y s q ac o ) a

e = 2, 4 and 8, wi h he
Mille indexes being included in he plo . In all he cases, h ee peaks ha index well o
a lamella lyo opic liquid c ys al phase (L

) we e obse ed, ega dless o

e , wi h he
cha ac e is ic in e -laye dis ance (d) di ec ly ela ed o he q ac o (d = 2πn/qhkl, n is
he di ac ion o de ). This s uc u e may be explained by conside ing ha he hexanoyl
chains linked o he wide en ance o he CD o us p omo e sel -assembling in a
lipidic bilaye ashion, CDplexes being adequa ely ep esen ed as al e na ing bilaye s o
paCDs molecules and an aqueous monolaye con aining supe coiled pDNA, wi h
hicknesses ep esen ed by dm, and dw, espec i ely, being d = dm + dw.
Table 3 collec s he alues o d, calcula ed as an a e age o he da a ob ained om
he mo e in ense peaks (100 and 200) o he di ac og ams a each

e a io. I is
no iceable ha he pe iodici y o he s uc u e emains basically unal e ed o a cons an
pDNA con en when he p opo ion o he ec o inc eases (i.e. d emains basically
cons an wi h

e ), o he h ee CDplexes s udied. Conside ing hese d alues and he
ac ha pDNA supe coils needs a ound dw ~2.0-2.5 nm o be sandwiched by paCDs
bilaye s in a ypical sandwich ashion, i can be deduced ha he hickness o he
bilaye (dm) mus be 3.1, 3.4 and 2.5 nm o ADM70-pDNA, ADM105-pDNA and
PBO234-pDNA CDplexes, espec i ely. Gi en he leng hs o bo h he ca ionic ails and
he lipid ype chains, and he dep h o he

-CD o us-shaped ca i y, hese d, dm and dw
alues a e compa ible wi h a L

s uc u e only i : i) he ca ionic ails on he na owe
en ance o he CD mac ocycle a e somehow in an open bouque ashion ( o a oid
elec os a ic epulsions among posi i e cha ges) and/o ii) he lipidic ype chains ha
a e linked o he wide en ance o -CD mac ocycle a e in e c ossed.
21
Fig. 3. SAXS di ac og ams o paCD-pDNA CDplexes a se e al e ec i e
cha ge a ios (ρe ): Red lines ρe = 2, g een lines ρe = 4, and blue lines ρe = 8
o : a) ADM70-pDNA; b) ADM105-pDNA; and c) PBO234-pDNA CDplexes.
1 2 3 4 5
(300)
(200)
(100)
q (nm-1)
(300)
(200)
(100)
c)
(300)
(200)
(100)
(300)
(200)
(100) a)
(300)
(200)
(100)
(300)
(200)
(100)
(300)
(200)
(100)
(100)
(100)
(300)
(200)
In ensi y (a.u.)
(300)
(200)
b)

22
Table 3. Values o q and d o he lamella (L

) liquid c ys al phase ound o he paCD-
pDNA CDplexes (whe e paCDs a e ADM70, ADM105 and PBO234), a se e al
e ec i e cha ge a ios (

e ).
ADM70
ADM105
PBO234

e
L

L

L

q100
1.12
1.06
1.28
2
q200
2.21
2.09
2.50
q300
3.30
3.12
-
d
5.6
5.9
5.0
q100
1.13
1.09
1.31
4
q200
2.23
2.13
2.47
q300
3.33
3.16
-
d
5.6
5.8
5.0
q100
1.15
1.07
1.30
8
q200
2.27
2.12
2.50
q300
3.39
3.18
-
d
5.5
5.9
4.9
Values o q and d a e epo ed in nm-1 and nm, espec i ely.
C yo-TEM expe imen s u he con i med he mul ilamella cha ac e o he
CDplexes s udied in his wo k. Fig. 4 shows a selec ion o mic og aphs among hose
aken o ADM105-pDNA CDplex ha a e ep esen a i e o he ensemble o da a (see
Fig. S1 in Supplemen a y da a o a selec ion o mic og aphs o ADM70-pDNA and
PBO234-pDNA CDplexes). The mul ilamella a angemen ound in SAXS is also seen
in hese mic og aphs. In ac , h ee ypes o nanos uc u es in coexis ence a e ound: (i)
CDplexes wi h a well-de ined mul ilamella p o ile and agg ega ed in a clus e ype
ashion (CT- ype nanoagg ega es; labelled wi h whi e as e isks in Fig. 4); (ii) CDplexes
wi h a clea inge -p in mul ilamella pa e n (FP- ype nanoagg ega es; labelled wi h
whi e ci cles, and a zoom iew in panel d), and; (iii) sel -agg ega ed paCDs wi hou
pDNA compac ed (labelled wi h whi e a ows), wi h a ypical esicle- ype s uc u e.
The p esence o agg ega es o paCD molecules, wi hou pDNA being compac ed, is
jus i ied by he ac ha he expe imen is done o samples wi h a clea excess o
ca ionic ec o (no ice ha

e = 4 in c yo-TEM expe imen s). These exceeding paCD
23
molecules end o o m unilamella sphe ical esicle- ype nanos uc u es, he o ma ion
o he lipidic bilaye being p omo ed by he p esence o 14 lipidic ype chains linked o
he wide en ance o each cyclodex in o us (2 chains pe glucose uni ). I can be
in e ed ha he unilamella paCD esicles abo e men ioned in e ac wi h pDNA
h ough s ong elec os a ic in e ac ions and end o agg ega e yielding he
mul ilamella phases, ei he CT o FP, by sandwiching he plasmid supe coils wi hin
he aqueous monolaye ha emain be ween each wo bilaye s. Scheme 2 shows
schema ic d awings o bo h nanoagg ega es: (a) unilamella esicles in he absence o
pDNA, and (b) mul ilamella complexes in he p esence o pDNA. In he CT- ype
s uc u es, he bilaye s may be de o med when sandwiching pDNA supe coils wi h he
adjacen bilaye s, bu hey essen ially keep hei mo phologies. Howe e , FP- ype
nanoagg ega es show he ypical inge -p in compac ion pa e n, and in con as wi h
he CT- ype CDplexes, he bilaye s end o dis up p obably due o a mo e a ou able
paCD-pDNA in e ac ion. This pa icula scena io has been also p e iously ound o
di e en lipoplexes (ca ionic lipids-pDNA complexes) [20,57]. Some o he
mul ilamella a angemen s shown in hese mic og aphs (and o he no shown) ha e
been chosen o analyze he p esence o pe iodici y. As ep esen a i e examples, he
inse s o panels b) and c) in Fig. 4, show Fas Fou ie T ans o m (FFT) p o iles whe e
he di ac ion spo co esponds o a ypical lamella pa e n.
24
Fig. 4. A selec ion o c yo-TEM mic og aphs showing a gene al iew o he ADM105-
pDNA CDplexes a

e = 4. Inse s on panels b) and c) show he di ac ion spo s om
FFT calcula ions o e a selec ed a ea on he o iginal mic og aph (yellow squa e). FFT
pa e n e eals a mul ilamella s uc u e. Scale ba s a e 100 nm in panels a-c and 50 nm
in panel d.
Scheme 2. Schema ic d awings o : a) Vesicles- ype paCD sel -agg ega ion pa e n; and
b) Mul ilamella lyo opic liquid c ys al phase (L

) o he CDplexes s udied in his
wo k, showing he s uc u al pa ame e s, d, dm and dw.
a)
b)
25
Acco dingly, he ensemble o elec ochemical and s uc u al esul s shows L sel -
assembling pa e ns i espec i ely o

e alues, while he bes compac ion le els a e
ob ained a

e = 4. Addi ionally, i is known ha CDplexes mus be posi i ely cha ged
(

e > 1), bu wi h ca ionic ec o con en being as low as possible o diminish
cy o oxici y. All hese conside a ions poin o

e = 4 as a po en ially adequa e cha ge
a io o ca y on bo h he p o eomic and TE s udies.
Upon in con ac wi h biological milieu, CDplexes will adso b plasma p o eins in a
ime-dependen manne . Mos abundan p o eins will bind o ec o su ace i s and
will be p og essi ely subs i u ed by p o eins wi h high a ini y o he su ace o
CDplexes. A he equilib ium ( ypically eached wi hin 1 h exposu e), he PC o
CDplexes will be cons i u ed by a longs anding p o ein laye e e ed o as he ha d
co ona (HC), which will p o ide CDplexes wi h hei biological iden i y, plus a so
co ona (SC) made o p o eins in dynamical exchange wi h he su ounding
en i onmen . In he p esen in es iga ion, bo h paCDs and CDplexes we e le o in e ac
wi h HP o 1 h. The PC o ma ion was con i med by means o

-po en ial and size
measu emen s o he CDplexes in he absence and in he p esence o HP. The da a
collec ed in Table 4 indica e ha he ba e complexes a e posi i ely cha ged (

-po en ial
a ound +30 mV), wi h mean hyd odynamic diame e s o a ound 114 nm in he case o
ADM70-pDNA CDplexes and a ound 150 nm o he o he wo CDplexes. A e 1 h
incuba ion wi h HP, a clea inc ease on he size o he complexes (hyd odynamic
diame e inc eases a ound 40-45 nm in he h ee cases), and a ma ked d op in

-
po en ial, shi ing om posi i e o nega i e ( om ca. +30 mV o ca. −17 mV), we e
obse ed. These e idences a e clea ly compa ible wi h he o ma ion o a ca. 20-nm-
hick PC, mos ly consis ing o nega i ely cha ged p o eins (i.e. pI < 7), ha a e loca ed
32
o p o ein exp ession, including endosome scape, a icking o he nucleus and pDNA
elease, a e pa icula ly a o able o PC-coa ed ADM70-pDNA CDplexes.
Fig.7. T ans ec ion e iciency (TE) alues o paCDs-pDNA CDplexes, in e ms o %
GFP exp essed (panels a-b) and mean luo escence in ensi y (MFI) (panels c-d), a

e =
4, 25 and 50, in he absence and p esence o Human Plasma (HP), h ough HeLa and
MCF-7 cells. CTR: cells alone. LFN: Lipo ec amine, posi i e con ol.
On he o he hand, as shown in Fig. 8, he inc ease on e ec i e cha ge a io (

e )
om 4 o 50 seems o sligh ly dec ease he iabili y o HeLa cells, in he absence o HP,
while no app eciable dec ease is obse ed in MCF-7 cells. In he p esence o PC, cell
iabili y o bo h cell lines was no app eciably diminished. No ice, none heless, ha
almos all he alues a e a ound o o e 80%, which is conside ed a minimum h eshold
alue. Howe e , in e p e a ion o he e ec o PC on he cell iabili y o CDplexes mus
be done wi h cau ion. Whe he PC educe cell iabili y o p o ec he cells can be due o
speci ic PC composi ion and, in u n, o he complex ela ionship be ween PC
composi ion and nanoca ie p ocessing by cell machine y [59]. Mo eo e , in he

33
absence o in i o expe imen s, he e alua ion o PC composi ion, al hough implies an
impo an imp o emen wi h espec o he use o SDS PAGE expe imen s alone, does
no allow one o depic s ong conclusions abou u u e in i o applica ions (i.e.
a ge ing abili y, bio-dis ibu ion, cy o oxici y). In ac , in he li e a u e, majo wo ks a e
discussing abou mapping p o ein binding si es ac oss he PC su ace. In he absence o
his piece o in o ma ion, discussion abou he po en ial ole o co ona p o eins should
be made, bu kep a a minimum, as done in his wo k. O he wise, he isk o
specula ion may be oo high.
Fig. 8. Cell iabili y o paCDS-pDNA CDplexes a

e = 4, 25 and 50 o HeLa and
MCF-7 cells, in he absence and p esence o human plasma (HP). CTR: cells alone.
CDS: cells in he p esence o paCDS wi hou pDNA.
The abo e p esen ed expe imen al e idences suppo ha he Janus paCD s uc u es
ADM70 and ADM105 a e p omising non i al ec o s o pDNA deli e y, o e all
34
supe io o PBO234 and o he Lipo ec amine con ol. Di e ences in ans ec ion
e iciency as a unc ion o he cell line migh a ise om di e ences in he p e e ed
in e naliza ion ou es. Thus, i has been epo ed ha CDplexes can en e he cell by
ca eolae- and cla h in-media ed endocy osis, bu only he i s mechanism is p oduc i e
ega ding p o ein exp ession [30]. Mo eo e , CDplexes ob ained om ADM70 seem o
exhibi he mos a ou able ea u es o in i o gene he apy applica ions: high s abili y
in he absence o p esence o HP, homogeneous size dis ibu ion, lowe impac (wi h
espec o he o he wo paCDs) o he PC in he capaci y o he CDplexes o e icien ly
media e p o ein exp ession in cellulo, and e y low cy o oxici y e en a high

e
alues, and independen ly o he p esence o no o HP. These ea u es a e in ag eemen
wi h he p e iously obse ed supe io i y o ADM70 o mula ions in in i o ans ec ion
s udies [33,34]. The body o biophysical and biochemical e idences ob ained in his
wo k p o ides a pa hway o he igo ous cha ac e iza ion o pDNA-molecula ec o
o mula ions in iew o hei op imiza ion o gene he apy applica ions. B oadening
he cu en da abase on ec o s uc u es, elec ochemical and s uc u al p ope ies o
hei agg ega es, p o ein co ona composi ion and ans ec ion e iciencies in di e en in
i o and in i o expe imen al se ings should und he basis o he a ional design o
second gene a ion candida es. Wo k in ha di ec ion is cu en ly sough in ou
labo a o ies.
4. Conclusions
This wo k was aimed o p o ide new insigh s in he ascina ing p ocesses go e ning
pDNA complexa ion by monodispe se ec o s o he Janus paCD amily, in an a emp
o delinea e he mechanisms whe eby di e ences in molecula s uc u e ansla es in o
di e ences in ans ec ion capabili ies. Fo ha pu pose, h ee Janus CD-based
35
compounds (ADM70, ADM105 and PBO234) ha e been ho oughly checked as
po en ial nanoca ie s o a pEGFP-C3 plasmid ha codes o GFP exp ession o he
in e io o HeLa and MCF-7 cance cells, bo h in he absence and p esence o human
se um. In a i s le el o o ganiza ion, he ec o molecula s uc u e in luences he
e ec i e posi i e cha ge a ailable o in e ac ing wi h he plasmid as well as he
e ec i e nega i e cha ge o he plasmid in he nanoagg ega es. In ac , ze a po en ial
s udy e ealed ha e ec i e cha ges a e lowe han nominal ones bo h o he CD-
based ec o s (a ound 30% o ADMs and 25% o PBO234), and also o he pDNA,
which ende s a low pe cen age o i s nega i e cha ge (less han 15%) when i is
compac ed by he nanoca ie s. This is a po en ially a ou able inding since he weake
he anionic cha ac e o he DNA, he lowe he amoun o ca ionic ec o needed o
o mula e he nanocomplexes, hus dec easing he cy o oxici y o he ec o and
inc easing hei po en ial ou pu as sa e and e ec i e ehicles o nucleic acids. On he
o he hand, SAXS and c yo-TEM s udies ha e shown ha ADM70-pDNA, ADM105-
pDNA and PBO234-pDNA CDplexes a e s uc u ed acco ding o a mul ilamella
lyo opic liquid c ys al phase (L

. Two di e en mul ilamella phases a e
dis inguished, none heless, on c yo-TEM mic og aphs (CT- ype and FP- ype
nanoagg ega es), in coexis ence wi h unilamella esicles o sel -agg ega ed exceeding
paCDs wi hou pDNA compac ed. Al hough he Janus ea u e seems o wa an a
mul ilamella a angemen o he paCD-pDNA nanocomplexes in all cases, hei
s abili y and homogenei y can be signi ican ly di e en depending on he opology o
he mul i alen ca ionizable domain in he paCD en i y. In he p esence o human
plasma, a second le el o o ganiza ion akes place in ol ing he in e ac ion wi h se um
p o eins, gi ing ise o mul icomponen nanoassemblies equipped wi h a p o ein co ona
whose composi ion a ies om a paCD o mula ion o ano he . The p o ein co ona
36
(PC) cha ac e iza ion has also d i en o in e es ing conclusions. The adso bed p o eins
ha e mos ly a nega i e cha ge in all he cases (pI < 7), poin ing o he elec os a ic
in e ac ions as he d i en non-co alen o ces be ween p o eins and he ca ionic ec o s.
Complemen , lipop o ein and coagula ion we e ound o be he mos abundan ypes o
p o eins wi hin he co ona o he Janus paCDs and he co esponding CDplexes,
whe eas immunoglobulins, issue leakage and acu e phase p o eins cons i u ed a mino
ac ion o he PC. A mode a e- o-high cha ge a ios (

e = 25 and 50), he ba e
CDplexes he ein epo ed seem o ans ec ei he HeLa o MCF-7 cance cells mo e
e icien ly han Lipo ec amine, and wi h high cell iabili ies anged om 80 o 100%.
These ans ec ion e iciencies we e ound o sligh ly dec ease in he p esence o
biological media (HP), bu in any case he alues ound we e highe han hose ob ained
wi h he con ol Lipo ec amine. Howe e , he e ec o PC on he cell iabili y o
CDplexes is no ha easy o in e p e . P obably, he speci ic PC composi ion and, in
u n, i s complex e ec on nanoca ie p ocessing by cell machine y, play a c ucial ole,
al hough an accu a e mapping o p o ein binding si es in he PC would be necessa y o
go u he in hese conclusions. In any case, he whole body o bo h biophysical and
biochemical e idences ob ained in his wo k allow us o conclude ha he h ee paCDs
p oposed can be conside ed as po en ially e icien nanoca ie s in i o and p omising
gen ec o s o in i o applica ions. Going u he , among he h ee Janus paCDS
nano ec o s checked in his wo k, ADM70 seem o exhibi he mos a ou able ea u es
o in i o gene he apy applica ions equi ing se um-con aining media, in o al
ag eemen wi h he al eady obse ed supe io i y o ADM70 o mula ions in in i o
ans ec ion s udies.
37
Acknowledgmen s
MINECO o Spain, (con ac numbe s CTQ2012-30821, SAF2013-44021-R and
CTQ2015-64425-C2-1-R), he Jun a de Andalucía (con ac numbe FQM2012-1467),
Uni e si y Complu ense o Mad id (Spain) (p ojec no. UCMA05-33-010) and he
Eu opean Regional De elopmen Funds (FEDER and FSE) o inancial suppo . SAXS
expe imen s we e pe o med a NCD11 beamline a ALBA Synch o on Ligh Facili y
wi h he collabo a ion o ALBA s a . Au ho s also hank C. Aica -Ramos o ca ying
on ampli ica ion o plasmid DNA a he Depa amen o de Bioquímica y Biología
Molecula I (UCM, Spain) and also P. Cas o-Ha mann, Se ei de Mic oscopia o UAB
(Spain), o c yo-TEM expe imen s.
Appendix A: Supplemen a y da a
Supplemen a y In o ma ion a ailable: Addi ional c yo-TEM mic og aphs, de ails and
ables o he op 25 mos abundan p o eins in he p o ein co ona o he CDplexes, and
addi ional TE and cell iabili y expe imen s a highe CDplexes concen a ions pe cell.
This in o ma ion can be ound on line a h p://dx.doi.o g/
Re e ences
[1] E. Junque a, E. Aica , Recen p og ess in gene he apy o deli e nucleic acids wi h
mul i alen ca ionic ec o s, Ad . Colloid In e ace Sci., 233 (2016) 161-175.
[2] I.M. Ve ma, M.D. Wei zman, Gene he apy: Twen y- i s cen u y medicine, Annu.
Re . Biochem., 74 (2005) 711-738.
[3] E. Junque a, E. Aica , Ca ionic lipids as ans ec ing agen s o DNA in gene
he apy, Cu . Topics Med. Chem., 14 (2014) 649-663.
[4] T. Mon ie , T. Ben egnu, P.A. Ja es, J.J. Yaouanc, P. Lehn, P og ess in ca ionic
lipid-media ed gene ans ec ion: A se ies o bio-inspi ed lipids as an example,
Cu . Gene The ., 8 (2008) 296-312.
[5] R.S. Dias, B. Lindman, DNA In e ac ion wi h Polyme s and Su ac an s, Wiley &
Sons, Hoboken, NJ, 2008.
[6] K. Ewe , N.L. Slack, A. Ahmad, H.M. E ans, A.J. Lin, C.E. Samuel, C.R. Sa inya,
Ca ionic lipid-DNA complexes o gene he apy: Unde s anding he ela ionship
be ween complex s uc u e and gene deli e y pa hways a he molecula le el, Cu .
Med. Chem., 11 (2004) 133-149.

38
[7] P.P. Ka mali, A. Chaudhu i, Ca ionic liposomes as non- i al ca ie s o gene
medicines: Resol ed issues, open ques ions, and u u e p omises, Med. Res. Re .,
27 (2007) 696-722.
[8] A.J. Ki by, P. Camille i, J. Engbe s, M.C. Fei e s, R.J.M. Nol e, O. Sode man, M.
Be gsma, P.C. Bell, M.L. Fielden, C.L.G. Rod iguez, P. Gueda , A. K eme , C.
McG ego , C. Pe in, G. Ronsin, M.C.P. an Eijk, Gemini su ac an s: New
syn he ic ec o s o gene ans ec ion, Angew. Chem., In . Ed., 42 (2003) 1448-
1457.
[9] P. Kesha wani, V. Gajbhiye, N.K. Jain, A e iew o nanoca ie s o he deli e y o
small in e e ing RNA, Bioma e ials, 33 (2012) 7138-7150.
[10] B. Khu ana, A.K. Goyal, A. Budhi aja, D. A o a, S.P. Vyas, siRNA deli e y using
nanoca ie s - An e icien ool o gene silencing, Cu . Gene The ., 10 (2010) 139-
155.
[11] M.A. Islam, T.E. Pa k, B. Singh, S. Maha jan, J. Fi dous, M.H. Cho, S.K. Kang,
C.H. Yun, Y.J. Choi, C.S. Cho, Majo deg adable polyca ions as ca ie s o DNA
and siRNA, J. Con olled Release, 193 (2014) 74-89.
[12] V.D. Badwaik, E. Aica , Y.A. Mondjinou, M.A. Johnson, V.D. Bowman, D.H.
Thompson, S uc u e-p ope y ela ionship o in i o siRNA deli e y pe o mance
o ca ionic 2-hyd oxyp opyl-β-cyclodex in: PEG-PPG-PEG poly o axane ec o s,
Bioma e ials, 84 (2016) 86-98.
[13] W. Kusse , Chemically modi ied nucleic acid ap ame s o in i o selec ions:
e ol ing e olu ion, Re . Molec. Bio echnol., 74 (2000) 27-38.
[14] B.E. Ea on, The joys o in i o selec ion: chemically d essing oligonucleo ides o
sa ia e p o ein a ge s, Cu . Opin. Chem. Biol., 1 (1997) 10-16.
[15] I.S. Blagb ough, A.A. Me wally, siRNA and Gene Fo mula ion o E icien Gene
The apy, InTech, 2013.
[16] C.E. Thomas, A. Eh ha d , M.A. Kay, P og ess and p oblems wi h he use o i al
ec o s o gene he apy, Na u e Re . Gen., 4 (2003) 346-358.
[17] P.D. Robbins, S.C. Ghi izzani, Vi al ec o s o gene he apy, Pha macol
The apeu ., 80 (1998) 35-47.
[18] M.A. Min ze , E.E. Simanek, Non i al ec o s o gene deli e y, Chem. Re ., 109
(2008) 259-302.
[19] K. Kuma , A.L. Ba an-Be don, S. Da a, M. Munoz-Ubeda, C. Aica -Ramos, P.
Kondaiah, E. Junque a, S. Bha acha ya, E. Aica , A delocalizable ca ionic
headg oup oge he wi h an oligo-oxye hylene space in gemini ca ionic lipids
imp o es hei biological ac i i y as ec o s o plasmid DNA, J. Ma e . Chem. B, 3
(2015) 1495-1506.
[20] A.L. Ba an-Be don, S.K. Mis a, S. Da a, M. Muñoz-Ubeda, P. Kondaiah, E.
Junque a, S. Bha acha ya, E. Aica , Ca ionic gemini lipids con aining
polyoxye hylene space s as imp o ed ans ec ing agen s o plasmid DNA in cance
cells, J. Ma e . Chem. B, 2 (2014) 4640-4652.
[21] D. Pu nam, Polyme s o gene deli e y ac oss leng h scales, Na u e Ma e ., 5
(2006) 439-451.
[22] D.J. Bha ali, I. Klejbo , E.K. S achowiak, P. Du a, I. Roy, N. Kau , E.J. Be gey,
P.N. P asad, M.K. S achowiak, O ganically modi ied silica nanopa icles: A
non i al ec o o in i o gene deli e y and exp ession in he b ain, PNAS, 102
(2005) 11539-11544.
[23] C. O iz Melle , J.M. Ga cia Fe nandez, J.M. Beni o, Glyco anspo e s o gene
deli e y, in: A.P. Rau e , T.K. Lindho s (Eds.) Ca bohyd a e Chemis y: Chemical
and Biological App oaches ol. 38, 2012, pp. 338-375.
39
[24] C. O iz Melle , J.M. Beni o, J.M. Ga cía Fe nandez, P eo ganized,
mac omolecula , gene-deli e y sys ems, Chem. Eu . J, 16 (2010) 6728-6742.
[25] Y. Aoyama, Mac ocyclic glycoclus e s: F om amphiphiles h ough nanopa icles o
glyco i uses, Chem. Eu . J, 10 (2004) 588-593.
[26] V. Bagnacani, V. F anceschi, M. Bassi, M. Lomazzi, G. Dono io, F. Sansone, A.
Casna i, R. Unga o, A ginine clus e ing on calix-4-a ene mac ocycles o imp o ed
cell pene a ion and DNA deli e y, Na u e Commun., 4 (2013).
[27] J.L. Jiménez Blanco, F. O ega-Caballe o, L. Blanco-Fe nández, T. Ca mona, G.
Ma celo, M. Ma ínez-Neg o, E. Aica , E. Junque a, F. Mendicu i, C. T os de
Ila duya, C. O iz Melle , J.M. Ga cía Fe nández, T ehalose-based Janus
cyclooligosaccha ides: he ‘‘click’’ syn hesis and DNA-di ec ed assembly in o pH-
sensi i e ans ec ious nanopa icles., Chem. Commun., 52 (2016) 10117-10120.
[28] S. Loe hen, J.M. Kim, D.H. Thompson, Biomedical applica ions o cyclodex in
based poly o axanes, Polyme Re iews, 47 (2007) 383-418.
[29] C. O iz Melle , J.M. Ga cía Fe nandez, J.M. Beni o, Cyclodex in-based gene
deli e y sys ems, Chem. Soc. Re ., 40 (2011) 1586-1608.
[30] A. Diaz-Moscoso, D. Ve cau e en, J. Rejman, J.M. Beni o, C. O iz Melle , S.C.
De Smed , J.M. Ga cía Fe nandez, Insigh s in cellula up ake mechanisms o
pDNA-polyca ionic amphiphilic cyclodex in nanopa icles (CDplexes), J.
Con olled Release, 143 (2010) 318-325.
[31] A. McMahon, E. Gomez, R. Donohue, D. Fo de, R. Da cy, C.M. O'D iscoll,
Cyclodex in gene ec o s: cell a icking and he in luence o lipophilic chain
leng h, J. D ug Deli . Sci. Technol., 18 (2008) 303-307.
[32] G. Ca acciolo, H. Ameni sch, Ca ionic liposome/DNA complexes: om s uc u e
o in e ac ions wi h cellula memb anes, Eu . Biophys. J., 41 (2012) 815-829.
[33] C. A anda, K. U biola, A.M. A doy, J.M. Ga cía Fe nandez, C. O iz Melle , C.
T os de Ila duya, Ta ge ed gene deli e y by new ola e-polyca ionic amphiphilic
cyclodex in-DNA nanocomplexes in i o and in i o, Eu . J. Pha m. Biopha m.,
85 (2013) 390-397.
[34] A. Mendez-A doy, K. U biola, C. A anda, C. O iz Melle , J.M. Ga cia Fe nandez,
C. T os de Ila duya, Polyca ionic amphiphilic cyclodex in-based nanopa icles o
he apeu ic gene deli e y, Nanomedicine, 6 (2011) 1697-1707.
[35] S.D. We ig, R.E. Ve all, M. Fold a i, Gemini su ac an s: A new amily o
building blocks o non- i al gene deli e y sys ems, Cu . Gene The ., 8 (2008) 9-
23.
[36] P.C. Bell, M. Be gsma, I.P. Dolbnya, W. B as, M.C.A. S ua , A.E. Rowan, M.C.
Fei e s, J. Engbe s, T ans ec ion media ed by gemini su ac an s: Enginee ed
escape om he endosomal compa men , J. Am. Chem. Soc., 125 (2003) 1551-
1558.
[37] A.L. Ba an-Be don, D. Pozzi, G. Ca acciolo, A.L. Cap io i, G. Ca uso, C.
Ca alie e, A. Riccioli, S. Palche i, A. Lagana, Time e olu ion o nanopa icle-
p o ein co ona in human plasma: Rele ance o a ge ed d ug deli e y, Langmui ,
29 (2013) 6485-6494.
[38] D. Walczyk, F.B. Bombelli, M.P. Monopoli, I. Lynch, K.A. Dawson, Wha he cell
“sees” in bionanoscience, J. Am. Chem. Soc., 132 (2010) 5761-5768.
[39] G. Ca acciolo, F. Ca da elli, D. Pozzi, F. Salomone, G. Macca i, G. Ba di, A.L.
Cap io i, C. Ca alie e, M. Papi, A. Lagana, Selec i e a ge ing capabili y acqui ed
wi h a p o ein co ona adso bed on he su ace o 1,2-dioleoyl-3-
ime hylammonium p opane/DNA nanopa icles, ACS Appl. Ma e . In e aces, 5
(2013) 13171-13179.
40
[40] G. Ca acciolo, S. Palche i, V. Colapicchioni, L. Digiacomo, D. Pozzi, A.L.
Cap io i, G. La Ba be a, A. Laganà, S eal h e ec o biomolecula co ona on
nanopa icle up ake by immune cells, Langmui , 31 (2015) 10764-10773.
1 M. und is , J. S igle , . Cede all, . e gg d, M. . lanagan, . ynch, .
Elia, K. Dawson, The e olu ion o he p o ein co ona a ound nanopa icles: a es
s udy, ACS Nano, 5 (2011) 7503-7509.
[42] A.L. Cap io i, G. Ca acciolo, C. Ca alie e, P. Foglia, D. Pozzi, R. Sampe i, A.
Lagana, Do plasma p o eins dis inguish be ween liposomes o a ying cha ge
densi y?, J. P o eomics, 75 (2012) 1924-1932.
[43] J.D. By ne, T. Be ancou , L. B annon-Peppas, Ac i e a ge ing schemes o
nanopa icle sys ems in cance he apeu ics, Ad . D ug Deli e y Re ., 60 (2008)
1615-1626.
[44] V. Mi sha iee, M. Mahmoudi, K. Lou, J. Cheng, M.L. K a , P o ein co ona
signi ican ly educes ac i e a ge ing yield, Chem. Commun., 49 (2013) 2557-2559.
[45] H. Ameni sch, G. Ca acciolo, P. Foglia, V. Fuscole i, P. Giansan i, C. Ma ianecci,
D. Pozzi, A. Lagana, Exis ence o hyb id s uc u es in ca ionic liposome/DNA
complexes e ealed by hei in e ac ion wi h plasma p o eins, Colloids Su . B, 82
(2011) 141-146.
[46] A.L. Cap io i, G. Ca acciolo, G. Ca uso, P. Foglia, D. Pozzi, R. Sampe i, A.
Lagana, DNA a ec s he composi ion o lipoplex p o ein co ona: A p o eomics
app oach, P o eomics, 11 (2011) 3349-3358.
[47] A. Diaz-Moscoso, L. Le Gou ie ec, M. Gomez-Ga cia, J.M. Beni o, P. Balbuena,
F. O ega-Caballe o, N. Guillo eau, C. Di Gio gio, P. Vie ling, J. De aye, C. O iz
Melle , J.M. Ga cía Fe nandez, Polyca ionic amphiphilic cyclodex ins o gene
deli e y: syn hesis and e ec o s uc u al modi ica ions on plasmid DNA complex
s abili y, cy o oxici y, and gene exp ession, Chem. Eu . J., 15 (2009) 12871-12888.
[48] A. Gadelle, J. De aye, Selek i e halogenie ung on cyclomal ooligosaccha iden in
C6‐posi ion und syn hese on Pe (3, 6‐anhyd o) cyclomal ooligosaccha iden,
Angew. Chem., 103 (1991) 94-95.
[49] M. Gómez-Ga cía, J.M. Beni o, D. Rod íguez-Lucena, J.-X. Yu, K. Chmu ski, C.
O iz Melle , R. Gu ié ez Gallego, A. Maes e, J. De aye, J.M. Ga cía Fe nández,
P obing seconda y ca bohyd a e-p o ein in e ac ions wi h highly dense
cyclodex in-cen e ed he e oglycoclus e s: he he e oclus e e ec , J. Am. Chem.
Soc., 127 (2005) 7970-7971.
[50] Y. Aoyama, T. Kanamo i, T. Nakai, T. Sasaki, S. Ho iuchi, S. Sando, T. Niidome,
A i icial i uses and hei applica ion o gene deli e y. Size-con olled gene
coa ing wi h glycoclus e nanopa icles, J. Am. Chem. Soc., 125 (2003) 3455-3457.
[51] S.K. Mis a, M. Muñoz-Ubeda, S. Da a, A.L. Ba an-Be don, C. Aica -Ramos, P.
Cas o-Ha mann, P. Kondaiah, E. Junque a, S. Bha acha ya, E. Aica , E ec s o a
delocalizable ca ion on he headg oup o gemini lipids on he lipoplex- ype nano-
agg ega es di ec ly o med om plasmid DNA, Biomac omolecules, 14 (2013)
3951-3963.
[52] M. Muñoz-Ubeda, S.K. Mis a, A.L. Ba an-Be don, S. Da a, C. Aica -Ramos, P.
Cas o-Ha mann, P. Kondaiah, E. Junque a, S. Bha acha ya, E. Aica , How does
he space leng h o ca ionic gemini lipids in luence he lipoplex o ma ion wi h
plasmid DNA? Physicochemical and biochemical cha ac e iza ions and hei
ele ance in gene he apy, Biomac omolecules, 13 (2012) 3926-3937.
[53] M. Muñoz-Ubeda, S.K. Mis a, A.L. Ba an-Be don, C. Aica -Ramos, M.B. Sie a,
J. Biswas, P. Kondaiah, E. Junque a, S. Bha acha ya, E. Aica , Why is less
41
ca ionic lipid equi ed o p epa e lipoplexes om plasmid DNA han linea DNA in
gene he apy?, J. Am. Chem. Soc., 133 (2011) 18014-18017.
[54] J. Bedna , C.L. Woodcock, Ch oma in, Academic P ess Inc, San Diego, CA, 1999.
[55] J. Duboche , M. Ad ian, J.J. Chang, J.C. Homo, J. Lepaul , A.W. McDowall, P.
Schul z, C yo-elec on mic oscopy o i i ied specimens, Q. Re . Biophys., 21
(1988) 129-228.
[56] J. Duboche , B. Zube , M. El so , C. Bouche -Ma quis, A. Al-Amoudi, F. Li olan ,
How o " ead" a i eous sec ion, in: Me hods Cell. Biol., ol. 79, 2007, pp. 385-
406.
[57] A.L. Ba an-Be don, M. Muñoz-Ubeda, C. Aica -Ramos, L. Pe ez, M.R. In an e,
P. Cas o-Ha mann, A. Ma in-Molina, E. Aica , E. Junque a, Ribbon- ype and
clus e - ype lipoplexes cons i u ed by a chi al lysine based ca ionic gemini lipid and
plasmid DNA, So Ma e , 8 (2012) 7368-7380.
[58] S. Palche i, D. Pozzi, C. Ma chini, A. Amici, C. And eani, C. Ba olacci, L.
Digiacomo, V. Gambini, F. Ca da elli, C. Di Rienzo, G. Pe uzzi, H. Ameni sch, R.
Pale mo, I. Sc epan i, G. Ca acciolo, Manipula ion o lipoplex concen a ion a he
cell su ace boos s ans ec ion e iciency in ha d- o- ans ec cells, Nanomed.
Nano echnol, Biol. Med., (2016) in p ess, DOI: 10.1016/j.nano.2016.1008.1019.
[59] D. Maiolo, P. Del Pino, P. Me angolo, W.J. Pa ak, F.B. Bombelli, Nanomedicine
deli e y: does p o ein co ona ou e o he a ge o o oad?, Nanomedicine, 10
(2015) 3231-3247.