Biophysics and protein corona analysis of Janus cyclodextrin-DNA nanocomplexes. Efficient cellular transfection on cancer cells

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. 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