Cationic nano-systems for DNA transfection

CATIONIC NANO-SYSTEMS FOR DNA TRANSFECTION B Y Diana Pa ícia Soa es de Pai a T HESIS SUBMITTED TO THE U NIVERSITY OF P ORTO FOR A D OCTOR OF P HILOSOPHY IN C HEMICAL AND B IOLOGICAL E NGINEERING S UPERVISION M ARIA DO C ARMO DA S ILVA P EREIRA S ANDRA C RISTINA P INTO DA R OCHA P ORTO , 2013 To my amily ... “Some people succeed because hey a e des ined o! Bu mos people succeed because hey a e de e mined o!” Hen y Fo d, 1863-1947 i Acknowledgmen s Acknowledgmen sAcknowledgmen s Acknowledgmen s This hesis and, he e o e, all he wo k done in he las 5 yea s was no possible wi hou he aluable con ibu ion o some people ha I am e y g a e ul. I hank o P o esso Ma ia do Ca mo Pe ei a o accep ing me as he PhD s uden in he Facul y o Enginee ing o he Uni e si y o Po o (FEUP, Po o, Po ugal) and o belie e on my capabili ies. He guidance, suppo , sugges ions and scien i ic discussions we e always essen ial. I would like o gi e my deepes app ecia ion o Doc o Sand a Rocha o he help and con ibu ion o his PhD. She has always iendly and hope ul, and made easie o o e come he di icul ies and us a ions o his wo k. Also, I exp ess my g a i ude o he membe s o ou in es iga ion g oup, pa icula ly, o P o esso Manuel Coelho, o he g oup scien i ic discussions and b ain s o ming. To LEPAE membe s and s a a special hank o hei help and con ibu ion. Some undamen al s udies whe e pe o med a Max Planck Ins i u e o Colloids and In e ace (Golm, Ge many), so I wan o hank o P o esso Helmu h Möhwald o he oppo uni y and o his hospi ali y. P o esso Ge ald B ezesinski dese es may deeply g a i ude o all he guidance and help ul discussions on Langmui monolaye s s udies and o being such a good ii and welcome iend. My s ay in Ge many was eally pleasan and I would like o hank all he iends I made he e, especially, P o esso Ge ald’s g oup o he an as ic wo king a mosphe e and my oomma es o being such a good iends. Many hanks o he co-wo ke s ha con ibu ed o his esea ch wo k. To Doc o Isabel Ca doso and he membe s o Molecula Neu obiology g oup om Ins i u o de Biologia Molecula e Celula (Po o, Po ugal), my app ecia ion o he help on cellula ans ec ion s udies. To Doc o Galya I ano a om Cen o de Ma e iais da Uni e sidade do Po o (Po o, Po ugal) o he help on NMR wo k. To Doc o Albe o Ma ín Molina and his g oup om Facul ad de Ciencias, Uni e sidad de G anada (G anada, Spain), o hei help on he liposome complexa ion heo y. To all my iends a Depa men o Chemical Enginee ing (FEUP, Po o, Po ugal) and Tuna Feminina de Engenha ia (TUNAFE, Po o, Po ugal) o he suppo , encou agemen and good imes o e he yea s. Thank you all o cons an p esence and iendship. To my amily, I o e all my g a i ude, o being he e and suppo ing my choices, encou aging me o pu sue my d eams and go u he . To my husband Joaquim F ei as a special hanks o being he e all he ime and helping me on he mos challenging p oblems. To my baby, Gab iel, hank you o colou ing my li e and make me smile. I am hank ul o Fundação pa a a Ciência e Tecnologia (FCT) o a PhD ellowship (SFRH/BD/45384/2008). The esea ch wo k was suppo ed by FCT esea ch p ojec s: Nano-NMed–SD/0156/2007 and PTDC/QUI- BIQ/102827/2008. iii Resumo ResumoResumo Resumo Es e abalho de in es igação isou a p epa ação e ca ac e ização de sis emas pa a o anspo e e en ega de ADN. Esses sis emas são ca iónicos e não i ais e são cons i uídos po lípidos e políme os. As monocamadas de Langmui o am u ilizadas pa a p e e as in e ações en e o lípido ca iónico DOTAP (Clo e o de ime il(2,3- dioleoilp opil)amónio) e o coles e ol (CHOL) ou o seu de i ado coles e ol luo ado (hep a luo ocoles e ol, F7-CHOL). A mis u a do DOTAP com cada um dos es e óis ( ácio 1:1) o igina monocamadas na ase líquida expandida, idên icas às do DOTAP. Ve i icou-se que a á ea po molécula das mis u as é mais pequena que a espe ada de aco do com a eg a da adição aplicá el no caso de ambos os compos os se em comple amen e miscí eis ou imiscí eis na monocamada. A adso ção de ADN na monocamada é simila pa a ambos os sis emas, o que sus en a a possibilidade da u ilização de coles e ol luo ado como lípido auxilia na o mulação de e o es pa a ans eção de ADN. A compac ação de ADN e espe i a ans eção o am es adas pa a lipossomas cons i uídos pelo DOTAP e pelo coles e ol luo ado ( ácio mola 1:1). Os esul ados con i ma am que mais lipossomas do sis ema DOTAP:F7- CHOL são necessá ios pa a compac a a mesma quan idade de ADN quando compa ado com o sis ema DOTAP:CHOL e que a ligação lipossoma-ADN é mais o e no caso do sis ema DOTAP:F7-CHOL/ADN. A es abilidade de ambos x 6. CHITOSAN-N-MALTODEXTRIN CONJUGATES................................................ 139 6.1. E XPERIMENTAL S ECTION ................................................................................ 140 6.1.1. C HEMICALS .................................................. ................................................... .. 140 6.1.2. S YNTHESIS OF CHITOSAN - GRAFT - MALTODEXTRIN .................................................. .. 140 6.1.3. F OURIER - TRANSFORM INFRARED (FTIR) SPECTROSCOPY .......................................... 141 6.1.4. N UCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY ........................................ 142 6.1.5. P REPARATION OF NANOPARTICLES .................................................. ...................... 144 6.1.6. P REPARATION OF POLYPLEXES .................................................. ............................ 145 6.1.7. DLS AND ZETA POTENTIAL MEASUREMENTS .................................................. ......... 145 6.1.8. T RANSMISSION ELECTRON MICROSCOPY (TEM) ............................................. ......... 146 6.1.9. S CANNING ELECTRON MICROSCOPY (SEM) ............................................. ............... 146 6.2. R ESULTS AND D ISCUSSION ............................................................................. 147 6.2.1. C HARACTERISATION OF CHITOSAN -N- MALTODEXTRIN COPOLYMER ............................ 147 6.2.2. C HITOSAN -N- MALTODEXTRIN NANOPARTICLES .................................................. ..... 152 6.2.3. C HITOSAN -N- MALTODEXTRIN /DNA COMPLEXES .................................................. .. 155 6.3. C ONCLUSIONS ............................................................................................. 157 6.4. R EFERENCES ............................................................................................... 158 7. CONCLUDING REMARKS ............................................................................. 163 LIST OF ABBREVIATIONS .................................................................................... 167 LIST OF FIGURES ................................................................................................ 175 LIST OF TABLES ................................................................................................. 179 xi P e ace P e aceP e ace P e ace Acco ding o he Law n° 216/92 o 13 h Oc obe and o he Doc o al Regula ions o he Uni e si y o Po o, we cla i y ha all he expe imen s, in e p e a ion and discussion p esen ed on his hesis a e ou own, excep i s a ed o he wise. In his disse a ion, esul s we e p esen ed om he ollowing publica ions: Pai a, D., Ma in-Molina, A., Ca doso, I., Quesada-Pe ez, M., Pe ei a, M. C., Rocha, S. (2013). “The e ec o a luo ina ed choles e ol de i a i e on he s abili y and physical p ope ies o ca ionic DNA ec o s”, So Ma e , 9, 401. Pai a, D., B ezesinski, G., Pe ei a, M. C., Rocha, S. (2013). “Langmui monolaye s o monoca ionic lipid mixed wi h choles e ol o luo ocholes e ol: DNA adso p ion s udies”, Langmui , 29, 1920. Pai a, D., Rocha, S., I ano a, G., Pe ei a, M. C. “Syn hesis and cha ac e iza ion o chi osan-mal odex in g a copolyme s: No el nanopa icles o d ug deli e y”, Physical Chemis y Chemical Physics, DOI: 10.1039/C3CP51215K. I NTRODUCTION 1 Chap e 1 1. 1.1. 1. In oduc ion In oduc ionIn oduc ion In oduc ion The gene he apy concep s a es ha a human disease migh be ea ed i he co ec gene ic ma e ial is supplied o he a ge ed cell. The new gene ic ma e ial can co ec o supplemen he de ec i e genes esponsible o he disease p og ession (Mansou i, 2004). Di e en gene he apeu ics o ea diseases such as cance , AIDS and neu ological diso de s a e cu en ly in clinical ials (Bi i, 2006; Ginn, 2013; Li, 2005; S aye , 2005). In gene he apy, he de elopmen and s udy o DNA ec o s wi h high ans ec ion e iciency and low oxici y is ex emely impo an . Ideally, a DNA deli e y sys em wi h he apeu ic pu poses should ha e high ans ec ion e iciency and high speci ici y o he a ge cell o educe he side e ec s. The sys em should in addi ion be small, s able, biodeg adable, easy o p epa e and should allow he DNA elease and exp ession (Mansou i, 2004; Pa il, 2005). Nanopa icles and nanocapsules o e many ad an ages in gene he apy due o hei e sa ile chemical s uc u es, su ace unc ionali ies and he abili y o ob ain con olled sizes. The ocus o his PhD esea ch wo k is o p epa e and cha ac e ise non- i al DNA deli e y sys ems based on lipids and polyme s. These sys ems a oid some o he p oblems associa ed o i al ec o s such as high oxici y 2 I NTRODUCTION and he gene a ion o a s ong immune esponse. O he ad an ages o non- i al ec o s a e easy o mula ion and assembly (Pa il, 2005). Non- i al ec o s a e ypically posi i ely cha ged such as ca ionic liposomes p epa ed wi h 1,2-dioleoyl-3- ime hylammonium-p opane, chlo ide sal (DOTAP) o ca ionic nanopa icles made o chi osan. These sys ems compac e icien ly he DNA h ough elec os a ic in e ac ions leading o he o ma ion o complexes (Mo ille, 2008). In his hesis he p epa a ion o h ee di e en non- i al sys ems is desc ibed. One sys em is based on luo ina ed compounds ( luo ina ed modi ied choles e ol) mixed wi h he ca ionic lipid DOTAP. Fluo ina ed ca ionic liposomes ha e shown a highe ans ec ion po en ial in in i o and in i o s udies when compa ed o hei analogues (K a , 2001). Also, hese compounds exhibi good p ope ies ega ding he anspo and p o ec ion o ac i e p inciples in he blood s eam (Riess, 2002). The use o luo ina ed choles e ol in ca ionic liposomes o DNA deli e y is desc ibed in he poin o iew o he helpe lipid e ec . Choles e ol is no mally added o lipid-based DNA ec o s o help he usion o he liposome wi h he memb ane, bu also o inc ease he s abili y and dec ease he oxici y o he sys em. The use o luo ina ed choles e ol migh inc ease u he he s abili y o he sys em due o i s high hyd ophobic cha ac e and p o ec DNA molecules om deg ada ion in he blood s eam, inc easing he hal -li e o he sys em (Boulange , 2004). A second ca ie was p epa ed wi h bolaamphiphiles, which a e molecules wi h wo hyd ophilic end g oups connec ed by a hyd ophobic ail. Bolaamphiphiles a e desc ibed o sel -assemble in wa e and o m packed monolaye lipid memb anes (Fo bes, 2006). A bolaamphiphile molecule wi h a hyd ophobic chain o 22 ca bon a oms, ime hylamine-qua e nized a one end and wi h a hyd oxyl g oup a he o he end was syn hesised. The ca ie s I NTRODUCTION 3 con aining his molecule we e p epa ed by wo di e en app oaches. One s a egy was o use he laye -by-laye echnique o assemble bolaamphiphile and DNA laye s on a mic opa icle co e ha could e en ually be emo ed o o igina e mic o- o nanocapsules (Sukho uko , 2007). A di e en app oach was o inco po a e he bolaamphiphile in esicles composed o he ca ionic lipid DOTAP. This mix u e is expec ed o o m s able esicles wi h small sizes e en in d y s a e due he wo pola heads and he long ca bon chain p esen in he bolaamphiphile (Jain, 2010). A DNA ec o sys em based on chi osan conjuga es is also p oposed. Chi osan is a non- oxic and na u al biodeg adable ca ionic polyme wi h low immunogenici y and high biocompa ibili y (Jayakuma , 2010). These p ope ies made chi osan one o he mo e used polyme s o gene he apies. Howe e , his molecule shows some p oblems, like solubili y a physiological pH and side e ec s as hypocholes e olemia i applied in high doses, limi ing i s applica ion. To o e come hese complica ions and o imp o e i s abili y o ans ec gene ic ma e ial, his ca ionic polyme was modi ied by g a ing a neu al polyme . Mal odex in was chosen because i is a non-ionic excipien ha enhances gene exp ession and has low oxici y (Huang, 2002). This hesis is o ganised in o se en chap e s. This chap e , in oduc ion, co e s he objec i es and scope o he p oposed wo k. Chap e 2, nano- sys ems o gene deli e y, con ains an o e iew o he non- i al ec o s conce ning hei applica ions in gene he apy and hei ad an ages and disad an ages. Chap e 3, in e ac ion o DNA wi h lipid monolaye s, co e s he use o Langmui lipid monolaye s o p edic he in e ac ions be ween he luo ina ed choles e ol and DOTAP and o s udy he in e ac ion o DNA molecules wi h he monolaye . Chap e 4, compac ion p ocess o DNA by luo ina ed liposomes, is dedica ed o he cha ac e isa ion o DOTAP liposomes mixed wi h luo ina ed choles e ol and he compa ison o his 4 I NTRODUCTION sys em wi h he well-known DOTAP:choles e ol liposomes. In chap e 5, bolaamphiphile-based ec o s, he po en ial applica ion o a new syn hesised bolaamphiphile in DNA ca ie s is discussed and in chap e 6, chi osan-N- mal odex in conjuga es, he syn hesis o chi osan-g a -mal odex in copolyme s, hei assembly in o nanopa icles and hei in e ac ion wi h DNA a e desc ibed. Finally, chap e 7, concluding ema ks, summa ises he main indings o his hesis. Re e ences Re e encesRe e ences Re e ences Bi i, A.; Capo ondo, A.; Fasano, S.; Ca o, U.d.; Ma chesini, S.; Azuma, H.; Malagu i, M.C.; Amadio, S.; B ambilla, R.; G ompe, M.; Bo dignon, C.; Qua ini, A.; Naldini, L. (2006). Gene he apy o me ach oma ic leukodys ophy e e ses neu ological damage and de ici s in mice, The Jou nal o Clinical In es iga ion, 116, 3070. Boulange , C.; Di Gio gio, C.; Gauche on, J.; Vie ling, P. (2004). T ans ec ion wi h luo ina ed lipoplexes based on new luo ina ed ca ionic lipids and in he p esence o a bile sal su ac an , Bioconjuga e Chemis y, 15, 901. Fo bes, C.C.; DiVi o io, K.M.; Smi h, B.D. (2006). Bolaamphiphiles P omo e Phospholipid T ansloca ion Ac oss Vesicle Memb anes, Jou nal o he Ame ican Chemical Socie y, 128, 9211. Ginn, S.L.; Alexande , I.E.; Edels ein, M.L.; Abedi, M.R.; Wixon, J. (2013). Gene he apy clinical ials wo ldwide o 2012 – an upda e, The Jou nal o Gene Medicine, 15, 65. Huang, C.-Y.; Ma, S.S.; Lee, S.; Radhak ishnan, R.; B aun, C.S.; Choosakoonk iang, S.; Wie ho , C.M.; Lobo, B.A.; Middaugh, C.R. (2002). Enhancemen s in gene exp ession by he choice o plasmid I NTRODUCTION 5 DNA o mula ions con aining neu al polyme ic excipien s, Jou nal o Pha maceu ical Sciences, 91, 1371. Jain, N.; A n z, Y.; Goldschmid , V. .; Dupo ail, G.; Mély, Y.; Klymchenko, A.S. (2010). New Unsymme ical Bolaamphiphiles: Syn hesis, Assembly wi h DNA, and Applica ion o Gene Deli e y, Bioconjuga e Chemis y, 21, 2110. Jayakuma , R.; Chennazhi, K.P.; Muzza elli, R.A.A.; Tamu a, H.; Nai , S.V.; Sel amu ugan, N. (2010). Chi osan conjuga ed DNA nanopa icles in gene he apy, Ca bohyd a e Polyme s, 79, 1. K a , M.P. (2001). Fluo oca bons and luo ina ed amphiphiles in d ug deli e y and biomedical esea ch, Ad anced D ug Deli e y Re iews, 47, 209. Li, C.; Bowles, D.E.; an Dyke, T.; Samulski, R.J. (2005). Adeno-associa ed i us ec o s: po en ial applica ions o cance gene he apy, Cance Gene The apy, 12, 913. Mansou i, S.; La igne, P.; Co si, K.; Bende dou , M.; Beaumon , E.; Fe nandes, J.C. (2004). Chi osan-DNA nanopa icles as non- i al ec o s in gene he apy: s a egies o imp o e ans ec ion e icacy, Eu opean Jou nal o Pha maceu ics and Biopha maceu ics, 57, 1. Mo ille, M.; Passi ani, C.; Vona bou g, A.; Cla eul, A.; Benoi , J.P. (2008). P og ess in de eloping ca ionic ec o s o non- i al sys emic gene he apy agains cance , Bioma e ials, 29, 3477. Pa il, S.; Rhodes, D.; Bu gess, D. (2005). DNA-based he apeu ics and DNA deli e y sys ems: A comp ehensi e e iew, The AAPS Jou nal, 7, E61. Riess, J.G. (2002). Blood subs i u es and o he po en ial biomedical applica ions o luo ina ed colloids, Jou nal o Fluo ine Chemis y, 114, 119. 6 I NTRODUCTION S aye , D.S.; Akkina, R.; Bunnell, B.A.; D opulic, B.; Planelles, V.; Pome an z, R.J.; Rossi, J.J.; Zaia, J.A. (2005). Cu en s a us o gene he apy s a egies o ea HIV/AIDS, Molecula The apy, 11, 823. Sukho uko , G.B.; Möhwald, H. (2007). Mul i unc ional ca go sys ems o bio echnology, T ends in Bio echnology, 25, 93. N ANO - SYSTEMS FOR GENE DELIVERY 7 Chap e 2 2. 2.2. 2. Nano NanoNano Nano- -- -sys ems o gene sys ems o gene sys ems o gene sys ems o gene deli e y deli e ydeli e y deli e y Human diseases migh be ea ed by he ans e o gene ic ma e ial in o speci ic cells in o de o co ec o supplemen de ec i e genes (Alde uccio, 2009; Buckley, 2011; Fische , 2010). Gene he apy is been s udied in clinical ials o di e en ype o diseases such as cance (B annon-Peppas, 2004; Li, 2005; Ramesh, 2001), HIV/AIDS (Kiem, 2012; S aye , 2005), diabe es (Callejas, 2013; Jean, 2011), cys ic ib osis (Mi omo, 2010; P ingle, 2009) and neu ological diso de s (Bi i, 2006; Kim, 2009; Man edsson, 2010). The e a e many p ocesses o pe o m gene he apy ea men s such as gene addi ion, gene co ec ion/al e a ion and gene knockdown ha could be used indi idually o in combina ion (Kay, 2011). Gene knockdown ac s by in oducing siRNA (sho in e e ing RNA) o miRNA (media ed gene egula ion ci cui s o RNA) o block o clea e a gene ansc ip , by selec ing he sequences ha should be eplica ed, elimina ing he de ec i e sequences (Summe on, 2007). Gene co ec ion/al e a ion is no a common echnique, howe e i is acqui ing some in e es by using a i icially enginee ed nucleases 14 N ANO - SYSTEMS FOR GENE DELIVERY Bo h liposomes and DNA possess nega i e cha ges, leading o a poo associa ion. The e o e no many s udies ha e been de eloped wi h his sys em o DNA deli e y. Howe e , i has been epo ed in he li e a u e ha DOPG (1,2-dioleoyl-sn-glyce o-3-[phospho- ac-(1-glyce ol)]) mixed wi h DOPE (1,2-dioleoyl-sn-glyce o-3-phosphoe hanolamine) can be complexed wi h DNA in he p esence o calcium ions (Ca 2+ ) and ans ec e ec i ely he DNA (Pa il, 2004; Pa il, 2005b). On he o he hand, ca ionic liposomes in e ac e icien ly wi h DNA molecules due o he opposi e cha ges. Ca ionic liposomes a e known o ha e mo e a ini y o he cell su ace o endo helial glycop o eins acili a ing he c osso e o he sys em h ough he memb ane (Rao, 2010). The e a e nume ous ype o ca ionic lipids used o gene deli e y (Geusens, 2011). DOTMA (1,2-di-O-oc adecenyl-3- ime hylammonium p opane) was he i s ca ionic lipid o be used. Cu en ly, i is used in se e al s udies and i is pa o he Lipo ec in® o mula ions, he i s comme cial o mula ion used as a s anda d o e alua e he e ec i eness o he new ans ec ion eagen s, despi e i s oxici y (Ta aho sky, 2009). DOTAP (1,2- dioleoyl-3- ime hylammonium-p opane, chlo ide sal ) is a mono alen ca ionic alipha ic lipid i s syn hesised by Le en is and Sil ius (Le en is, 1990). I has a high po en ial o ans ec ion s udies combined wi h i s low oxici y, becoming one o he mo e used ca ionic lipids in gene he apy s udies (Ca acciolo, 2012). O he ca ionic lipids also used a e DC-Chol (3β-[N-(N',N'- dime hylaminoe hane)-ca bamoyl]choles e ol hyd ochlo ide) (Ca acciolo, 2005; Muñoz-Úbeda, 2010); mul i alen alipha ic lipids such as DOGS (dioc adecylaminoglycylspe mine) and DOSPA (2,3 Dioleyloxy-N- [2(spe mineca boxaminino)e hyl]-N,N-dime hyl-1-p opanaminium i lu oace a e). DOSPA is one o he lipids p esen in Lipo ec amine®, one o N ANO - SYSTEMS FOR GENE DELIVERY 15 he mo e used comme cial o mula ions a ailable as eagen o assess ans ec ion in cell expe imen s (Ped oso de Lima, 2003). The ans ec ion e iciency o ca ionic liposomes is dependen on he lipid molecule, he p esence o helpe lipids, he lipid/DNA a io and which cells a e a ge ed. Helpe lipids, like choles e ol o DOPE (dioleoyl phosphoe hanolamine), a e no mally neu al in cha ge and a e used o imp o e he sys em s abili y, educe he oxici y o ca ionic lipids and inc ease he hal -li e o liposomes and hei ans ec ion e iciency in in i o s udies (C ook, 1998; Hui, 1996; Miguel, 2003; Xu, 2008; Zhang, 2004). To imp o e he o mula ions based on ca ionic lipids, a new gene a ion o lipids modi ied wi h luo ine a oms has eme ged. Fluo ine is he mos elec onega i e o he elemen s and i has a high ionisa ion po en ial and a e y low pola izabili y (Kissa, 1994). The eby, liquid luo oca bons p esen low an de Waals in e ac ions and low cohesi e ene gy densi ies (Riess, 1994a). Consequen ly hese luo oca bons show e y low su ace ensions, excellen sp eading p ope ies, high luidi y, low dielec ic cons an and high comp essibili y (K a , 1998). The la ge su ace exhibi ed by hese luo ina ed chains combined wi h he low pola izabili y esul s on enhanced hyd ophobici y. They show bo h hyd ophobic and lipophobic p ope ies a he same ime (K a , 2001; Riess, 2009). Many luo oca bons a e biocompa ible, e en a la ge doses hey appea o be innocuous and physiologically inac i e. No oxici y, ca cinogenic, mu agenic e ec s o immunological eac ions ha e been desc ibed o pu e luo oca bons wi h a molecula weigh anging om 460 o 520 g/mol. A phase I clinical ial e ealed ha pe luo ooc yl b omide emulsion a a dosage o 1.2 o 1.8 g o luo oca bon/kg do no p esen side e ec s in heal hy humans (K a , 2001; Leese, 2000; Riess, 1984). 16 N ANO - SYSTEMS FOR GENE DELIVERY Fluo ina ed amphiphiles ha e inc eased endency o sel -assemble in wa e when compa ed o hei hyd ogena ed analogues due o he s ong hyd ophobic cha ac e o he luo ina ed chains (K a , 2003). They also ha e a lowe c i ical micella concen a ion han he hyd ogena ed analogues (Shinoda, 1972). Fluo ina ed lipids ha e been syn hesised and es ed as he main ca ionic lipid o DNA ec o s (analogues o ca ionic DOSPA) as well as he helpe lipid (analogues o DOPE) (Gauche on, 2000; Gauche on, 2001a; Gauche on, 2001b; Gauche on, 2002; Klein, 2010; O mane Boussi , 2001). They ha e shown ad an ages o e hei hyd ogena ed analogues and ca ionic polyme s (polye hylenimine) ega ding he ans ec ion po en ial (Huh, 1996). Due o he s ong endency o sel -assemble, luo ina ed lipids p e en he in e ac ion o DNA wi h lipophilic o hyd ophilic molecules esponsible o lipo ec ion inhibi ion, and p o ec DNA om deg ada ion in he bloods eam since hemoly ic ac i i y is s ongly supp essed (Boulange , 2004; Riess, 1991; Riess, 1994b). Bolaamphiphile lipids di e om con en ional lipids because hey ha e wo hyd ophilic heads in opposi e sides o he molecule ins ead o one. These head g oups a e connec ed h ough a hyd oca bon chain (Fuh hop, 1986). The mo e known na u al bolaamphiphile is he monolaye ed memb ane o he mophilic and acidophilic a chaebac e ia, as Sul olobus sol a a icus o Sul olobus acidocalda ius species (Baek, 2010; Rosa, 1986). These molecules exhibi p omising applica ions in gene he apy (Dakwa , 2012; Nu aje, 2012). They ha e enhanced physical s abili y due o he p esence o he wo pola g oups. These molecules can be symme ical o asymme ical wi h espec o he cha ge p esen on he head g oups. I is possible o syn hesise a molecule wi h he same head g oup a bo h ends o wi h a posi i ely cha ged g oup a one end and a neu al g oup a he o he . Acco ding o he cha ges, he molecules will ea ange o o m s able esicles N ANO - SYSTEMS FOR GENE DELIVERY 17 ha could be used o encapsula e, anspo and deli e an ac i e p inciple (B unelle, 2009; G inbe g, 2010; Kau man, 2013). The sel -assembly o bolaamphiphiles in o s uc u es can be e y di e en , depending on i s cha ac e is ics. I he bolaamphiphile has symme ical head g oups, i will ha e a endency o gene a e monolaye ed esicles. Howe e i he head g oups a e asymme ical, he ea angemen could be e y di e en . Usually, hey o m monolaye lipid memb anes when he head g oups a e smalle o abou o he same size o he hyd ophobic co e. Fo bigge head g oups, he molecules could o m bilaye ed s uc u es ha a e mo e igid and can ha e c ys al shapes. In c ys al assemblies, h ee ypes o a angemen s can occu . Conside ing a and b he head g oups o an asymme ical bolaamphiphile, he bilaye could be assembled in a pa allel way, whe e he a head g oup is o ien ed o he inne space and he b head g oups o he ou e space o ice- e sa wi h espec o he head g oups (Figu e 2.2.a/b). This o ien a ion will depend on he o ces p esen on he head g oups and in he solu ion bulk o su ace. These bolaamphiphiles can also o ganise hemsel es in an an ipa allel way, whe e he a head g oup is o ien ed o a b head g oup, o ming in he inne space a sequence o a-b-a in one laye o he co esponding sequence b-a-b on he o he (Figu e 2.2.c) (Fuh hop, 2004). Despi e he in luence o he head g oups on he assembly o hese s uc u es, he hyd ophobic pa o he molecule also plays an impo an ole on he way ha hese molecules sel -assemble. Long ca bon chains usually lead o monolaye ed lipid memb anes, simila o liposomes, while sho hyd oca bon chains gene a e esicles like micelles (Jain, 2010; Jain, 2012; Popo , 2010). The p esence o double bonds be ween ca bons o o he g oups in he hyd ophobic chain con e s igidi y o he molecule and could lead o a U-shaped o ien a ion o he molecule in which he hyd ophilic 18 N ANO - SYSTEMS FOR GENE DELIVERY g oups a e aligned on he same side and he U bend is in he opposi e side, gene a ing a bilaye ed esicle (Meis e , 2007; Yan, 2009). Mix u es o bolaamphiphile wi h lipids a e used o imp o e he p ope ies o he anspo ec o (Casche a, 2011). The lipids p esen assembled acco ding a bilaye esicle and he bolaamphiphile can be in e cala ed i he size o i s hyd ophobic pa is close o wice o he size o he hyd ophobic pa o he lipid molecule (Hal e , 2004). Bolaamphiphile molecules can also acqui e he U-shape o be e blend wi hin he bilaye (Gu, 2003; Hu e , 2012; Moss, 1991). Figu e 2.2. Schema ic ep esen a ion o possible a angemen s o asymme ical bolamphiphiles in c ys als: a) pa allel a,a; b) pa allel a,b; c) an ipa allel a,b and b,a (Fuh hop, 2004). N ANO - SYSTEMS FOR GENE DELIVERY 19 2.2.2. 2.2.2.2.2.2. 2.2.2. Polyme ic Polyme ic Polyme ic Polyme ic ca ie s ca ie sca ie s ca ie s Polyme ic gene ec o s a e p epa ed by complexa ion o ca ionic polyme s wi h anionic DNA molecules gene a ing nanosphe es wi h diame e s anging om 50 o 700 nm (Pannie , 2004). Polyplexes o polyme -DNA complexes a e in e nalised by cells h ough endocy osis o memb ane usion, due o he o e all posi i e cha ge o he complex, and he DNA elease happens upon endosomal escape by p o on-sponge mechanism (Boussi , 1995; Jewell, 2008). These ec o s a e mo e s able and less oxic han liposome sys ems and condense mo e DNA (Mansou i, 2004). O he ad an ages o hese sys ems include low immunogenici y, e sa ili y o physicochemical p ope ies and easy manu ac u e (Me dan, 2002). Ca ionic polyme s ypically in e ac wi h DNA in a s onge way, h ough elec os a ic in e ac ions, leading o he o ma ion o complexes wi h mul iple DNA molecules. The e o e he size o he pa icles is ela ed mos ly wi h he polyme physical p ope ies han he DNA molecule size (Min ze , 2009). Howe e , biocompa ibili y and, p incipally, ans ec ion e iciency s ill ha e o be imp o ed. Polyme ec o s s ongly depend on he na u e o he polyme and hei molecula weigh and on he ionic s eng h o he solu ions. Modi ica ions on he polyme s uc u e con e speci ic physiological and physicochemical p ope ies ha help wi h he DNA up ake and he issue a ge ing (Me dan, 2002). Polye hylenimine (PEI) is one o he mo e used polyme s in gene he apeu ics. I can be syn hesised in di e en leng hs, be b anched o linea and can be unc ionalised o be e compa ibili y. Rega ding polyme ec o s, PEI p esen s good esul s when i comes o ans ec ion e iciency (Pun, 2004). I condenses DNA e icien ly o ming homogeneous sphe ical nanopa icles. 20 N ANO - SYSTEMS FOR GENE DELIVERY B anched PEI has a highe molecula weigh being mo e cy o oxic, bu also condenses mo e DNA since i possesses mo e a ailable posi i e cha ges, o ming smalle nanopa icles. Linea PEI wi h low molecula weigh shows highe ans ec ion e iciency alues and lowe cy o oxici y when compa ed o b anched PEI (Pa k, 2006). Despi e i s aluable cha ac e is ics, PEI showed high oxici y in in i o ials, mos ly due o he high amoun o posi i e cha ges and he non-biodeg adabili y o he compound. In o de o educe i s oxici y, s udies we e pe o med o g a o he polyme s like polye hylene glycol (PEG), a non-ionic hyd ophilic polyme (Pe e sen, 2002) o β-cyclodex in (β- CD), a suga polyme wi h low oxici y (Pun, 2004). Poly(L-lysine) (PLL) is a homopolypep ide ha combined wi h i s biodeg adable na u e make i an ideal polyme o DNA ans ec ion. PLL wi h a molecula weigh abo e 3 kDa can e ec i ely bind DNA molecules o o m s able complexes. Howe e , a physiological pH, all p ima y amine g oups o PLL a e p o ona ed, which hampe s he elease o DNA by he endosomes and, he e o e, has a weak ans ec ion e iciency. In addi ion, PLL complexes exhibi a high cy o oxici y (Mo ille, 2008). To o e come hese p oblems, a nume ous o biodeg adable PLL conjuga es ha e been syn hesised. Poly(lac ic- co-glycolic acid)(PLGA)-g a ed-PLL conjuga es showed educed oxici y and enhancemen s on he ans ec ion e iciency (Jeong, 2002). Es e -linked PLL- PEG mul iblock copolyme s combined wi h a ious a ios o his idine esidues p omo e gene ans e and educe he cy o oxici y when compa ed o he non-de i a ised PLL (Bik am, 2004). Polylysine has non-speci ic cell a ge ing. De i a isa ion o PLL molecules wi h a ge ing moie ies is one o he ecu en solu ions o his p oblem. Hepa ic cell a ge ing uses galac ose (Han, 2000) o lac ose (Choi, 1998) moie ies. Tumo issue a ge ing was achie ed by conjuga ing PLL o ola e (Cho, 2005) o ans e in (Wagne , 1991) molecules. N ANO - SYSTEMS FOR GENE DELIVERY 21 In o de o a ge leukaemia T-cells a speci ic JL1 an igen was coupled o polylysine (Suh, 2001). Na u al polysaccha ides o ca bohyd a es a e used in gene he apy due o hei p ope ies such as biodeg adabili y, educed oxici y, long hal -li e in he blood s eam and he abili y o a ge cells o he li e , spleen, lung and spinal-co d (Liu, 2008). Nume ous saccha ides a e used in gene deli e y such as dex an (Bish , 2009), amylose (Kaneko, 2007) and cyclodex in (Teijei o- Oso io, 2009). Nanopa icles o chi osan and i s de i a i es a e s udied in he pha maceu ical ield as d ug ca ie s and DNA deli e y sys ems (Pannie , 2004; Pa el, 2012). Chi osan is a linea polyme o β-(1-4)-linked-D- glucosamine monome s wi h andomly dis ibu ed N-ace yl-D-glucosamine uni s ob ained by pa ial deace yla ion o chi in, a na u al polysaccha ide ound p incipally in c us acean shells such as sh imps and c abs (Jayakuma , 2010b). I is a non- oxic and na u al biodeg adable ca ionic polyme wi h low immunogenici y and high biocompa ibili y. O he ad an ages o his mac omolecule include s abili y, e sa ili y o physicochemical p ope ies and he possibili y o modi ica ion by a aching cell compa ible ligands (Jayakuma , 2010a). Howe e , p oblems ela ed o chi osan deli e y sys ems ha e been epo ed: he pH dependence (chi osan is insoluble a neu al pH); he con ol o pa icle size, shape and polydispe si y due o he a iabili y o chi osan molecula weigh ; he limi a ion o chi osan applica ion in humans due o hypocholes e olemia caused by high doses o chi osan; he in i o ans ec ion a e o DNA-chi osan nanopa icles is e y much dependen on he cell ype (Maso i, 2009; Pa il, 2005a; Sa o, 2001). The sys ems a e ypically p epa ed ei he by complexa ion o he ca ionic polyme wi h anionic DNA o by encapsula ion/en apmen o he ac i e p inciple in he chi osan ma ix. Chi osan colloidal sys ems ha e 22 N ANO - SYSTEMS FOR GENE DELIVERY no mally a size anging om a ew nanome es o 500 nm (Agniho i, 2004; Fel , 1998; Liu, 2008; Nguyen, 2009). They ha e been used in non- con en ional d ug deli e y o nasal, ocula and pe o al adminis a ion in o de o p olong he con ac ime and p o ec DNA om nuclease deg ada ion (Mansou i, 2004; Sa anya, 2011). The e a e many ac o s in luencing he gene exp ession o d ug deli e y using chi osan sys ems (Kim, 2007; Mao, 2010). Kiang e al. es ed chi osan molecules wi h di e en molecula weigh and deg ee o deace yla ion and de e mined ha hese p ope ies play a key ole in he op imisa ion o he o mula ions o DNA ans ec ion (Kiang, 2004). An al e na i e app oach o imp o e chi osan p ope ies is o chemically modi y i s s uc u e by g a ing o he molecules (Dünnhaup , 2012; Gao, 2008; Kaneko, 2007; Zhou, 2011). Ma hew e al. conjuga ed olic acid wi h ca boxyme hyl chi osan o p oduce nanopa icles capable o a ge ing and con olling he elease o 5- luo ou acil, an an icance d ug used in chemo he apy (Ma hew, 2010). Pa k e al. sugges ed g a ing dex an o galac osyla ed chi osan o a ge ing genes o hepa ocy es (Pa k, 2001). Unmodi ied chi osan was also g a ed o dex an molecules o di e en molecula weigh s, esul ing in compounds wi h di e en deg ees o subs i u ion and wa e solubili y a di e en pH (Janciauskai e, 2008). Dend ime s a e sphe ical, highly b anched polyme s. They consis o a cen al co e molecule, whe e highly b anched a ms g ow o o m a ee-like s uc u e (Chen, 2000). The ami ica ion happens in a well-o de ed and symme ical way wi h he epe i ion o he same sequence o eac ions, p oducing monodispe se nanopa icles. Dend ime s a e classi ied by he numbe o gene a ions, which co esponds o he numbe o epea ed b anching sequences ha a e pe o med du ing he syn hesis. High gene a ion dend ime s (10) ha e supe io ans ec ion e iciencies when N ANO - SYSTEMS FOR GENE DELIVERY 23 compa ed o low gene a ion dend ime s (2) (Zhu, 2010). Polyamidoamine (PAMAM) is one o he mo e popula dend ime s. I is sa e, non-immunogenic and has a high capaci y ega ding ans ec ion e iciency, due he p esence o p ima y and e ia y amines (Vaidya, 2011). The p ima y amine g oups play an impo an ole on he DNA binding, p oducing compac nanopa icles and p omo ing he cellula up ake. The e ia y amines a ou he elease o DNA in o he cy oplasm by disabling he endosomes (Vaidya, 2011). 2.2.3. 2.2.3.2.2.3. 2.2.3. Ino ganic Ino ganicIno ganic Ino ganic based basedbased based nanopa icles nanopa iclesnanopa icles nanopa icles Apa om liposomal and polyme ic ec o s, some new DNA ehicles ha e gained a en ion in he las ew yea s. Ino ganic nanopa icles allies op ical, magne ic and o he physical p ope ies o he ine ness, s abili y and ease o unc ionaliza ion, making hem an a ac i e al e na i e o o ganic ehicles (Huang, 2011). Me al based nanopa icles hold a high po en ial o applica ions in bo h diagnos ic imaging and a ge ed d ug deli e y. No mally, hese nanopa icles a e deli e ed in colloidal o mula ions wi h inc eased ci cula o y hal -li e and ca y la ge amoun s o d ug due o i s ex emely small sizes wi h la ge su ace a eas (Naahidi, 2013). Gold nanopa icles (AuNPs) a e one o he mo e s udied me als as deli e y sys em. They a e easy o syn hesise wi h di e en sizes, and he p ocess is simple, cheap and eliable. S udies ha e shown ha hese nanopa icles a e biocompa ible, non- oxic and ha hei nega i e cha ged su ace can be easily unc ionalised wi h di e se mac omolecules (Li, 2009; Pa een, 2012). Bha a ai e al. modi ied he su ace o gold nanopa icles wi h chi osan. This o mula ion o med complexes wi h plasmid DNA and exhibi ed highe ans ec ion e iciency in he s omach and in es ine a e o al deli e y, when compa ed wi h Lipo ec in® o mula ion (Bha a ai, 2008). Li e al. coa ed gold nanopa icles 30 N ANO - SYSTEMS FOR GENE DELIVERY wi h Fluo ina ed Glyce ophosphoe hanolamine Helpe Lipids, Bioconjuga e Chemis y, 12, 949. 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Nu aje, N.; Bai, H.; Su, K. (2012). Bolaamphiphilic molecules: Assembly and applica ions, P og ess in Polyme Science, O mane Boussi ; Jé ôme Gauche on; Ca oline Boulange ; Ca he ine San aella; Hanno V. J. Kolbe; Pie e Vie ling (2001). Enhanced in i o and in i o ca ionic lipid-media ed gene deli e y wi h a luo ina ed glyce ophosphoe hanolamine helpe lipid, The Jou nal o Gene Medicine, 3, 109. Pannie , A.K.; Shea, L.D. (2004). Con olled elease sys ems o DNA deli e y, Molecula The apy, 10, 19. 38 N ANO - SYSTEMS FOR GENE DELIVERY Pa k, I.K.; Pa k, Y.H.; Shin, B.A.; Choi, E.S.; Kim, Y.R.; Akaike, T.; Cho, C.S. (2001). Galac osyla ed chi osan-g a -dex an as hepa ocy e- a ge ing DNA ca ie , Jou nal o Con olled Release, 75, 433. Pa k, T.G.; Jeong, J.H.; Kim, S.W. (2006). Cu en s a us o polyme ic gene deli e y sys ems, Ad anced D ug Deli e y Re iews, 58, 467. Pa een, S.; Mis a, R.; Sahoo, S.K. (2012). 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Syn hesis, Cha ac e iza ion, and Biocompa ibili y o Polye hylenimine-g a - poly(e hylene glycol) Block Copolyme s, Mac omolecules, 35, 6867. Popo , M.; Linde , C.; Deckelbaum, R.J.; G inbe g, S.; Hansen, I.H.; Shaubi, E.; Wane , T.; Heldman, E. (2010). Ca ionic esicles om no el bolaamphiphilic compounds, Jou nal o Liposome Resea ch, 20, 147. N ANO - SYSTEMS FOR GENE DELIVERY 39 P ingle, I.; Hyde, S.; Gill, D. (2009). Non- i al ec o s in cys ic ib osis gene he apy: ecen de elopmen s and u u e p ospec s, Expe Opinion on Biological The apy, 9, 991. Pun, S.H.; Bellocq, N.C.; Liu, A.; Jensen, G.; Macheme , T.; Quijano, E.; Schluep, T.; Wen, S.; Engle , H.; Heidel, J.; Da is, M.E. (2004). Cyclodex in- Modi ied Polye hylenimine Polyme s o Gene Deli e y, Bioconjuga e Chemis y, 15, 831. Ramesh, R.; Saeki, T.; Temple on, N.S.; Ji, L.; S ephens, L.C.; I o, I.; Wilson, D.R.; Wu, Z.; B anch, C.D.; Minna, J.D.; Ro h, J.A. (2001). Success ul ea men o p ima y and dissemina ed human lung cance s by sys emic deli e y o umo supp esso genes using an imp o ed liposome ec o , Molecula The apy, 3, 337. Rao, N.M. (2010). Ca ionic lipid-media ed nucleic acid deli e y: beyond being ca ionic, Chemis y and Physics o Lipids, 163, 245. Riess, J.G. (1984). Reassessmen o C i e ia o he Selec ion o Pe luo ochemicals o Second-Gene a ion Blood Subs i u es: Analysis o S uc u e/P ope y Rela ionships, A i icial O gans, 8, 44. Riess, J.G.; Pace, S.; Za i , L. (1991). Highly e ec i e su ac an s wi h low hemoly ic ac i i y, Ad anced Ma e ials, 3, 249. Riess, J.G. (1994a). Highly luo ina ed sys ems o oxygen anspo , diagnosis and d ug deli e y, Colloids and Su aces A: Physicochemical and Enginee ing Aspec s, 84, 33. Riess, J.G. (1994b). Fluo ina ed Vesicles, Jou nal o D ug Ta ge ing, 2, 455. Riess, J.G. (2009). Highly luo ina ed amphiphilic molecules and sel - assemblies wi h biomedical po en ial, Cu en Opinion in Colloid & In e ace Science, 14, 294. Rod igues, C.; Gamei o, P.; Reis, S.; Lima, J.L.; de Cas o, B. (2001). De i a i e spec opho ome y as a ool o he de e mina ion o d ug pa i ion 46 I NTERACTION OF DNA WITH LIPID MONOLAYERS conside able knowledge on in e ac ions be ween di e en species, hei molecula o ien a ion and de ec s, he shape and ex u e o he domains and he wo-dimensional la ice s uc u e. These echniques a e also used o mimic he binding o polyelec oly es o he in e ace (pep ides, p o eins, DNA) and o simula e enzyma ic eac ions a he memb ane su aces (B ezesinski, 2003). The s udy using Langmui monolaye s p o ided in o ma ion abou he lipid mix u es, DOTAP and choles e ol o luo ina ed choles e ol ha was use ul o he s udies wi h liposomes. A compa a i e s udy be ween choles e ol and luo ina ed choles e ol as he helpe lipids in DOTAP sys ems was pe o med using Langmui monolaye s udies (G omelski, 2006; Symie z, 2004). P essu e-a ea iso he ms, su ace po en ial and B ews e angle mic oscopy measu emen s we e ca ied ou o gain in o ma ion abou molecula dis ibu ion and mixing beha iou o he lipids in he monolaye . The DNA adso p ion p ocess a he monolaye in e ace was s udied by ib a ional spec oscopy (B ezesinski, 2003). DNA is expec ed o adso b om he subphase o he in e ace due o elec os a ic o ces and o induce changes in he densi y and composi ion o he in e ace laye , which is de ec ed by in a ed e lec ion abso p ion spec oscopy (G omelski, 2006). 3.1. 3.1.3.1. 3.1. Lipid Lipid Lipid Lipid Monolaye expe imen s Monolaye expe imen sMonolaye expe imen s Monolaye expe imen s 3.1.1. 3.1.1.3.1.1. 3.1.1. Chemicals ChemicalsChemicals Chemicals DOTAP (1,2-dioleoyl-3- ime hylammonium-p opane, chlo ide sal , MW 698.542), choles e ol (o ine wool, > 98%, MW 386.355) and F7- choles e ol (25,26,26,26,27,27,27-hep a luo ocholes e ol, MW 512.587) (Figu e 3.1) we e pu chased om A an i Pola Lipids, Inc. Salmon es es DNA I NTERACTION OF DNA WITH LIPID MONOLAYERS 47 and PBS (phospha e bu e ed saline: 0.01 M phospha e bu e , 0.0027 M po assium chlo ide and 0.137 M sodium chlo ide, pH 7.4) we e pu chased om Sigma-Ald ich. The deionised wa e was pu i ied wi h Milli-Q appa a us wi h he speci ic esis ance o 18.2 MΩ∙cm. Chlo o o m (Me ck) was used o dissol e he lipids. 3.1.2. 3.1.2.3.1.2. 3.1.2. P P P P essu e/a ea Iso he ms essu e/a ea Iso he msessu e/a ea Iso he ms essu e/a ea Iso he ms The ea ly esea ch on Langmui monolaye s s a ed ha di e en wo- dimensional phases exis o co-exis . Monolaye phases a e classi ied in gaseous (G), Liquid-expanded (LE), Liquid-condensed (LC) and solid (S), as isible in Figu e 3.2. The monolaye comp essibili y a ies acco ding o he sequence G>LE>LC>S, and i is de e mined by measu ing he su ace p essu e- DOTAP Choles e ol Hep a luo ocholes e ol Figu e 3.1. Chemical s uc u es o DOTAP, CHOL (choles e ol) and F7-CHOL (hep a luo ocholes e ol). 48 I NTERACTION OF DNA WITH LIPID MONOLAYERS molecula a ea (π-A) iso he ms unde equilib ium condi ions (Vollha d , 2006). The la e al su ace p essu e π o he monolaye is he di e ence be ween he su ace ension (γ) o pu e wa e (72.8 mN/m a 20°C) and wa e co e ed wi h monolaye (G omelski, 2004; 2006): = − (3.1) The su ace p essu e is a measu emen o he cohesi e ene gy p esen a he in e ace. The lipid molecules will in e ac wi h each o he h ough a ac i e and epulsi e o ces (F ei e, 2007). The ai /wa e in e ace possesses an excess o ee ene gy om he di e ence in en i onmen be ween he su ace molecules and hose in he bulk. This in e acial ee ene gy is esponsible o he su ace ension. The su ace p essu e-a ea iso he ms we e eco ded using a PTFE Langmui ilm balance sys em (Riegle & Ki s ein, Po sdam, Ge many), ep esen ed on Figu e 3.3. Figu e 3.2. Rep esen a i e phases o a su ace p essu e-a ea iso he m o a Langmui monolaye . I NTERACTION OF DNA WITH LIPID MONOLAYERS 49 Monolaye s o DOTAP, choles e ol, F7-choles e ol o hei mix u es we e ob ained by sp eading he solu ion o lipids in chlo o o m a a concen a ion o 1 mM on he bu e su ace (PBS bu e o PBS con aining 0.1 mg/mL o DNA). The su ace ension was de e mined wi h a con inuous Wilhelmy- ype p essu e measu ing sys em using a 10 mm wide il e pape as pla e. The monolaye s we e comp essed a a cons an eloci y o 5 Å 2 /molecule/min and a a empe a u e o (20 ± 1)°C. The a e age a ea pe molecule A o an ideal mix u e was calcula ed by he addi i i y ela ionship conside ing he mola ac ion X o each componen , A = X 1 A 1 + X 2 A 2 (3.2) 3.1.3. 3.1.3.3.1.3. 3.1.3. Su ace Po en ial Su ace Po en ialSu ace Po en ial Su ace Po en ial Cells need an elec ic ield and concen a ion p o iles o ions in he p oximi y o hei memb anes o exis . These pa ame e s a e desc ibed by he classical Gouy-Chapman (GC) model o elec ical double laye (EDL). Howe e Figu e 3.3. Langmui ilm balance sys em. 50 I NTERACTION OF DNA WITH LIPID MONOLAYERS de ia ions om ha ule happen in non-homogeneously cha ged su aces, like he lipid wa e in e ace o many mix u es (Shapo alo , 2006). Su ace po en ial expe imen s p o ide addi ional in o ma ion on he packing and o ien a ion o he lipid molecules a he monolaye . The po en ial o he su ace is ead as a unc ion o he a ea pe molecule and depends on he monolaye phase. A la ge a eas pe molecule he su ace po en ial is close o ze o. I emains cons an ill a c i ical a ea is eached on comp ession, inc easing o dec easing depending on he cha ge o he lipids in s udy (Oli ei a, 1997). These c i ical a eas no mally coincide wi h he inc ease o he su ace p essu e alues on Langmui iso he ms, indica ing ha he monolaye become mo e s uc u ed han be o e, due he comp ession e ec . Su ace po en ial measu emen s we e eco ded using a homemade de ice (V. L. Shapo alo , Ins i u e o Chemical Physics, RAS, Moscow, Russia) ha combines a Kel in p obe senso and a Wilhelmy pla e in o de o eco d a he same ime he su ace po en ial and he su ace p essu e iso he m. B ie ly, a 250 x 100 x 8 mm 3 Te lon ough wi h a single ba ie (asymme ic comp ession) con ains a gold-pla ed elec ode o 10 mm diame e ha ib a es a ca. 80 Hz. The e e ence elec ode was made o Ag/AgCl (Shapo alo , 2006). Su ace po en ial and su ace p essu e was se o ze o be o e sp eading he monolaye on o he subphase and he esul s a e eco ded a he same condi ions desc ibed abo e. Final su ace po en ial da a we e e e enced o he su ace po en ial o a subphase wi hou he monolaye . 3.1.4. 3.1.4.3.1.4. 3.1.4. B ews e Angle Mic oscopy (BAM) B ews e Angle Mic oscopy (BAM)B ews e Angle Mic oscopy (BAM) B ews e Angle Mic oscopy (BAM) BAM echnique was i s de eloped o cla i y he s uc u al cha ac e is ics o amphiphilic subs ances a he ai -wa e in e ace. I p o ides in o ma ion on he mo phology o he monolaye s including he I NTERACTION OF DNA WITH LIPID MONOLAYERS 51 inne s uc u e o he o med domains as well as he e ogenei ies in hin ilms (Melze , 1997). Inhomogenei ies on he monolaye can be om lipid densi y a ia ion a he wa e su ace (Symie z, 2004). A clean and pe ec in e ace be ween wo media wi h di e en e ac i e index has no e lec ion i i is illumina ed by pa allel (p)-pola ised ligh unde a unique angle o incidence, called B ews e angle (α). Fo he ai /wa e in e ace, he B ews e angle is 53.1° (Henon, 1991; Kae che , 1993). The o ma ion o a hin ilm wi h a di e en e ac i e index changes he op ical s a us ha is now e lec ing he ligh and p oducing an image o he monolaye (Rod íguez Pa ino, 1999). The occu ence o domains wi h highe molecula densi ies will e lec he ligh and be isible as b igh e a eas compa ed wi h he da k backg ound o he wa e su ace. The sys em used is a ailable comme cially (BAM2, Nano ilm Technology L d., Ge many; 20 mW lase , wa eleng h 514 nm, esolu ion 3 μm) and consis s o a CCD came a ha de ec s he p-pola ised ligh o he diode lase e lec ed om he su ace o ilm cha ac e isa ion. The B ews e angle o ai -wa e in e ace is 53.1°. The eco ded images we e analysed wi h he exis ing so wa e and co ec ed o he pe spec i e o obse a ion unde he B ews e angle wi h a spa ial esolu ion o abou 2 µm. 3.1.5. 3.1.5.3.1.5. 3.1.5. In In In In a ed Re lec ion Abso p ion a ed Re lec ion Abso p ion a ed Re lec ion Abso p ion a ed Re lec ion Abso p ion Spec oscopy (IRRAS) Spec oscopy (IRRAS)Spec oscopy (IRRAS) Spec oscopy (IRRAS) In a ed e lec ion abso p ion spec oscopy o lipid monolaye s a he ai /wa e in e ace con ibu es wi h unique molecula s uc u e and o ien a ion in o ma ion om he ilm compounds. Pa ame e s as lipid chain con o ma ion, il angle and p esence o DNA molecules a he in e ace could 52 I NTERACTION OF DNA WITH LIPID MONOLAYERS be de e mined wi h his echnique ia changes in ib a ional equencies (B ezesinski, 2003; G omelski, 2004). IRRAS is based on he obse a ion ha when mid-IR adia ion eaches he aqueous monolaye s, a ac ion o he ligh is e lec ed om he molecula compounds o he hin ilm a he su ace. Ligh in a well-de ined pola isa ion s a e, ei he pa allel (p-pola ised) o pe pendicula (s-pola ised) o he plane o incidence, impinges on o he su ace a a well-de ined and con olled angle o incidence, which is e lec ed and de ec ed a he same angle (Mendelsohn, 2010). Table 3.1 shows some o he mo e impo an Mid-IR g oup equencies o phospholipids and DNA (Banyay, 2003; Blume, 1996; G omelski, 2006). Wa enumbe (cm-1) Symbol Desc ip ion DNA cha ac e is ic bands 1720-1710 ν(C=O) DNA base pai ing 1225-1220 νas(PO2 - ) Asymme ic PO2 - s e ching (B o m) 1090-1085 νs(PO2 - ) Symme ic PO2 - s e ching o backbone (A/B o m) 1069-1044 ν(C-O) C-O s e ching o backbone 970 ν(C-C) C-C s e ching o backbone (B o m) Phospholipid cha ac e is ic bands 3400-3700 ν(O-H) H-O-H s e ching 2925 νas(CH2) Asymme ic CH2 s e ching (LE phase) 2918 νas(CH2) Asymme ic CH2 s e ching (LC phase) 2855 νs(CH2) Symme ic CH2 s e ching (LE phase) 2849 νs(CH2) Symme ic CH2 s e ching (LC phase) 1740 ν(C=O) C=O s e ching Table 3.1. Mo e common Mid-IR g oup equencies o phospholipids and DNA (Banyay, 2003; Blume, 1996; G omelski, 2006). I NTERACTION OF DNA WITH LIPID MONOLAYERS 53 IRRAS spec a we e eco ded using an IFS 66 FTIR spec ome e (B uke , Ge many) equipped wi h a liquid ni ogen cooled MCT de ec o . The spec ome e was coupled o a Langmui ilm balance, placed in a sealed con aine , o gua an ee a cons an wa e apou a mosphe e. The IR beam was ocused on he wa e su ace o he Langmui ough a an angle o incidence equal o 40° and was pola ised e ically (s) using a KRS-5 ( hallium b omide and iodide mixed c ys al) wi e g id pola ise . FTIR spec a we e collec ed a 8 cm -1 esolu ion wi h 400 scans and sub ac ed om he spec um o he e e ence ough (PBS bu e ). The wai ing ime o DNA adso p ion was he same in all expe imen s and was 1 hou (G omelski, 2006). 3.2. 3.2.3.2. 3.2. Resul s and Discussion Resul s and DiscussionResul s and Discussion Resul s and Discussion 3.2.1. 3.2.1.3.2.1. 3.2.1. DOTAP/F7 DOTAP/F7DOTAP/F7 DOTAP/F7- -- -choles e ol monolaye s choles e ol monolaye scholes e ol monolaye s choles e ol monolaye s The su ace p essu e-a ea (π-A) iso he m o DOTAP on phospha e bu e ed saline (PBS) is cha ac e is ic o a liquid-expanded phase and has an ini ial p essu e inc ease a 105 Å 2 /molecule. The addi ion o choles e ol (CHOL) o F7-choles e ol (F7-CHOL) o DOTAP (chemical s uc u es shown in Figu e 3.1) a 1:1 a io leads o he same o e all liquid-expanded phase beha iou wi h a dec ease in he a ea pe molecule (Figu e 3.4). The iso he ms o he pu e CHOL monolaye and he pu e F7-CHOL monolaye on PBS bu e a e iden ical and a e cha ac e is ic o a condensed phase, showing small changes in a e age molecula a ea wi h inc easing su ace p essu e. The ini ial inc ease is a 55 Å 2 /molecule and hei limi ing a ea A 0 (ex apola ion o he linea pa o he iso he m o π = 0) is 40 Å 2 /molecule. The la e al in e ac ions be ween DOTAP and he s e ols we e analysed by calcula ing he 54 I NTERACTION OF DNA WITH LIPID MONOLAYERS a e age a ea pe molecule wi hin he mixed monolaye as a unc ion o lipid concen a ion a he su ace p essu es o 5 and 35 mN/m (Table 3.2). A nega i e de ia ion o he ideal mixing o comple e demixing is obse ed expe imen ally, pa icula ly a 5 mN/m, suppo ing he condensing e ec o he s e ols. BAM analysis o DOTAP/CHOL 1:1 shows domains a su ace p essu es below 15 mN/m, which disappea comple ely when he p essu e eaches 20 mN/m (Figu e 3.4). 20 40 60 80 100 120 140 160 0 5 10 15 20 25 30 35 DOTAP/F7-CHOL CHOL F7-CHOL Su ace p essu e (mN/m) A ea pe molecule (Å 2 ) DOTAP DOTAP/ F7-CHOL on DNA DOTAP/F7-CHOL π = 0.5 mN/m (65 Å2 ) π = 0 mN/m ( 90 Å 2 ) DOTAP/CHOL π = 20 mN/m (47 Å 2 ) DOTAP/CHOL π = 10 mN/m (52 Å 2 ) Figu e 3.4. P essu e-a ea iso he ms o monolaye s o DOTAP, choles e ol (CHOL), F7-choles e ol (F7-CHOL), DOTAP/choles e ol 1:1 mix u e (DOTAP/CHOL) and DOTAP/F7-choles e ol 1:1 mix u e (DOTAP/F7-CHOL) on PBS bu e and o DOTAP/F7-choles e ol 1:1 mix u e on 0.1 mg/mL o DNA dissol ed in PBS. B ews e angle mic oscopy images o he mix u es o DOTAP and he s e ols a di e en su ace p essu es a e also shown. I NTERACTION OF DNA WITH LIPID MONOLAYERS 55 The domains ha e well-de ined shapes. DOTAP/F7-CHOL mix u e o ms domains only a π = 0 mN/m, demons a ing ha hei miscibili y is no comple e, bu he monolaye is homogeneous immedia ely when he su ace p essu e is inc eased (Figu e 3.4). In he case o DOTAP/CHOL 1:1 monolaye a demixing is obse ed indica ing a miscibili y gap be ween wo liquid phases: liquid-diso de ed wi h mo e DOTAP and liquid-o de ed wi h mo e CHOL. The iso he ms a e no sensi i e enough o dis inguish be ween hese wo phases because he di e ence in he molecula a ea mus be e y small and will e en dec ease on inc easing p essu e because he uppe c i ical miscibili y poin will be app oached. This is a phase diag am e y simila o he one obse ed o 1,2-dimy is oyl-sn-glyce o-3-phosphocholine (DMPC)/CHOL sys em (Okonogi, 2004; Wagne , 2008). In he case o DOTAP/F7-CHOL he e is only a liquid-o de ed phase. Coupling o DNA o DOTAP o mix u es o DOTAP and he s e ols (1:1 a io) leads o he expansion o he monolaye s, indica ing a pa ial pene a ion o he DNA (iso he m shown o he DOTAP/F7-CHOL case, Figu e Lipid Monolaye Am (Å 2 ) Calcula ed Am (Å 2 ) ∆ ∆∆ ∆ V (mV) π = 5 mN/m 35 mN/m 5 mN/m 35 mN/m 35 mN/m DOTAP 88.3 57.7 - - + 560 CHOL 39.8 35.3 - - + 430 DOTAP/CHOL 1:1 57.2 42.0 64.1 46.5 + 540 F7-CHOL 40.5 36.4 - - − 620 DOTAP/F7-CHOL 1:1 55.6 42.3 64.4 47.1 + 20 Table 3.2. A e age molecula a ea (Am) and su ace po en ial (∆ ∆∆ ∆V) o he lipid monolaye s a cons an su ace p essu es. 62 I NTERACTION OF DNA WITH LIPID MONOLAYERS CHOL has he CH 2 symme ic s e ching band a 2850 cm -1 , indica ing an all- ans con o ma ion (condensed phase). The same is alid o F7-CHOL CH2 s e ching bands. The CH 2 asymme ic s e ching band is no conside ed in his case since i con ains signi ican o e lapping con ibu ions om me hyl g oups (Lewis, 1998). In he case o pu e CHOL and F7-CHOL monolaye s, DNA is no de ec ed a he in e ace. F om di ec obse a ion o he in ensi y o he symme ic s e ching band o he phospha e g oup (1088 cm -1 ) o DNA i is possible o pe o m a compa ison o he di e en sys ems. When he amoun o posi i ely cha ged lipids dec eases, a dec easing in he amoun o abso bed DNA is isible. This dec ease o he band in ensi y is mo e no iceable o he mix u e o DOTAP and F7-CHOL. The di e ence in OH-band in ensi ies a ound 3600 cm -1 be ween DOTAP, DOTAP/CHOL and DOTAP/F7-CHOL also indica es a smalle amoun o adso bed DNA o he mix u e wi h he luo ina ed choles e ol. Lipid Monolaye on PBS o DNA/PBS νsCH2 (cm - 1 ) π=5 mN/m 35 mN/m DOTAP 2854 2854 CHOL 2850 2850 DOTAP/CHOL 1:1 2854 2854 F7-CHOL 2850 2850 DOTAP/F7-CHOL 1:1 2854 2854 Table 3.3. Symme ic CH2 ib a ion band o he lipid monolaye s ob ained by IRRAS. I NTERACTION OF DNA WITH LIPID MONOLAYERS 63 3.3. 3.3.3.3. 3.3. Conclusion ConclusionConclusion Conclusions ss s The use o F7-CHOL as he helpe lipid in ca ionic non- i al gene ec o s is p oposed. The e ec o his neu al lipid on he s uc u e o monolaye s o med by he ca ionic lipid DOTAP was s udied and compa ed wi h ha o CHOL. Bo h iso he ms and IRRAS esul s demons a e ha he mix u es o DOTAP and he s e ols (1:1 mola a io) a e in he liquid expanded s a e. The mix u es appea ed homogeneous a p essu es be ween 30-35 mN/m, which is conside ed he la e al p essu e in liposomes and will ha e implica ions o he abili y o he lipoplexes o med by hese lipids o condense DNA. F7-CHOL and CHOL induce a condensing e ec on DOTAP monolaye s bu BAM showed ha CHOL is less miscible wi h DOTAP han F7- CHOL. CHOL and F7-CHOL o ien simila ly in a e ical way a he in e ace h oughou comp ession wi h he OH g oup owa ds he pola in e ace, as concluded om he su ace po en ial measu emen s and molecula a eas. F om IRRAS esul s, i is possible o conclude ha a smalle amoun o DNA is adso bed a DOTAP/F7-CHOL monolaye s when compa ed wi h DOTAP/CHOL. Since he iso he ms o he mix u es show e y simila molecula a eas, simila cha ge densi ies a e expec ed in he mixed monolaye s. The only possible explana ion o he momen is a di e en miscibili y beha iou be ween DOTAP and CHOL o F7-CHOL, espec i ely. I CHOL is less miscible wi h DOTAP, mo e pa ches con aining di e en amoun s o CHOL and he e o e di e en cha ge densi ies a e expec ed. This is in line wi h he BAM expe imen s showing a homogeneous DOTAP/F7-CHOL monolaye a any la e al p essu e abo e ze o. This leads o a homogeneous monolaye wi h a smalle cha ge densi y compa ed o ha o he DOTAP/CHOL mix u es, which shows he coexis ence o choles e ol- ich and choles e ol-poo domains up o 20 mN/m, indica ing he coexis ence o pa ches wi h highe o smalle cha ge 64 I NTERACTION OF DNA WITH LIPID MONOLAYERS densi ies in he mixed monolaye . The pa ches wi h he highe cha ge densi y a e esponsible o he a ac ion o a la ge amoun o DNA. Acco ding o hese esul s, F7-CHOL and DOTAP a e comple ely miscible, and DNA adso bs o his mixed monolaye e en a ionic s eng h close o he physiological alues, hus F7-CHOL is a p omising candida e o he p epa a ion o gene deli e y ec o s. 3.4. 3.4.3.4. 3.4. 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(2010). In a ed e lec ion–abso p ion spec oscopy: P inciples and applica ions o lipid–p o ein in e ac ion in Langmui ilms, Biochimica e Biophysica Ac a (BBA) - Biomemb anes, 1798, 788. Mille , A.; Helm, C.A.; Mohwald, H. (1987). The Colloidal Na u e o Phospholipid Monolaye s, Jou nal De Physique, 48, 693. Möhwald, H., Phospholipid Monolaye s, in Handbook o Biological Physics R.L.a.E. Sackmann, Edi o . 1995, Else ie Science The Ne he lands. p. 161. O'Hagan, D. (2008). Unde s anding o gano luo ine chemis y. An in oduc ion o he C-F bond, Chemical Socie y Re iews, 37, 308. Okonogi, T.M.; McConnell, H.M. (2004). Con as in e sion in he epi luo escence o choles e ol-phospholipid monolaye s, Biophysical Jou nal, 86, 880. Oli ei a, O.N.; Bona di, C. (1997). The su ace po en ial o Langmui monolaye s e isi ed, Langmui , 13, 5920. Rod íguez Pa ino, J.M.; Sánchez, C.C.; Rod íguez Niño, M.R. (1999). 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P og ess in cha ac e iza ion o Langmui monolaye s by conside a ion o comp essibili y, Ad ances in Colloid and In e ace Science, 127, 83. I NTERACTION OF DNA WITH LIPID MONOLAYERS 69 Wagne , K.; Desba , B.; B ezesinski, G. (2008). Liquid-liquid immiscibili y in model memb anes ac i a es sec e o y phospholipase A2, Biochimica E Biophysica Ac a, 1778, 166. Zuidam, N.J.; Ba enholz, Y. (1997). Elec os a ic pa ame e s o ca ionic liposomes commonly used o gene deli e y as de e mined by 4- hep adecyl-7-hyd oxycouma in, Biochimica E Biophysica Ac a- Biomemb anes, 1329, 211. C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES 71 Chap e 4 4. 4.4. 4. Compac ion p ocess o DNA Compac ion p ocess o DNA Compac ion p ocess o DNA Compac ion p ocess o DNA by luo ina ed liposomes by luo ina ed liposomesby luo ina ed liposomes by luo ina ed liposomes Nume ous ca ionic lipids ha e been syn hesised and es ed o gene deli e y in cell cul u es, animal models and pa ien s who ake pa in clinical ials (Mo ille, 2008; Zhu, 2010). Gene he apy uses plasmid DNA o cellula RNA in e e ence machine y o u n o gene exp ession in cance cells o o he diseased cells (Aagaa d, 2007; Mo ille, 2008). The use o naked nucleic acids is limi ed by he p esence o se um nucleases and hus i is only alid o di ec injec ion o he mac omolecule on issues ha a e easily accessible such as skin and muscle (Mo ille, 2008). These lipids a e usually used o gene deli e y in he o m o liposomes ha a e complexed wi h DNA h ough elec os a ic in e ac ions. The oxici y o hese complexes, named lipoplexes, is ela ed o he cha ge a io be ween he ca ionic liposomes and DNA plus he dose ha is adminis e ed (L , 2006). Also, he oxici y o such sys ems in cell cul u es is a ibu ed o he ee liposomes p esen in he mix u e, as he sepa a e componen s o he lipoplexes do no show any oxici y when injec ed in mice models (Xu, 1999). Thus he s udy o he in e ac ion be ween 78 C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES wi h maximum luo escence in ensi y o he exci a ion spec um and ice e sa (Lakowicz, 2006). Fluo escence measu emen s a e commonly execu ed in s eady-s a e bu ime- esol ed measu emen s can also be done. S eady-s a e measu emen s a e pe o med wi h cons an illumina ion o he sample by a con inuous beam o ligh and he emission spec um is eco ded due o he nanosecond scale o luo escence (Lakowicz, 2006). S eady-s a e luo escence measu emen s we e ca ied ou using a Pe kin-Elme LS-50B luminescence spec ome e (REQUIMTE, Depa men o Chemis y, Facul y o Pha macy, Uni e si y o Po o). The DNA concen a ion was kep cons an (0.025 mg/mL) and liposomes we e added a liposome- DNA mass a io (L/D) a ying om 0 o 10, simila ly o he o he cha ac e isa ion s udies. E hidium b omide (E B ) was added o DNA solu ion a a DNA:E B mass a io o 6:1. Fluo escence emission spec a we e eco ded be ween 560-700 nm a an exci a ion wa eleng h o 540 nm, emission and exci a ion band sli s o 10 nm and scan a e o 400 nm/min. All spec a we e sub ac ed o he spec a o blanks (bu e o bu e plus liposomes a he same concen a ions ha we e used o he o ma ion o lipoplexes). 4.1.6. 4.1.6.4.1.6. 4.1.6. T ansmission Elec on Mic oscopy T ansmission Elec on Mic oscopyT ansmission Elec on Mic oscopy T ansmission Elec on Mic oscopy T ansmission elec on mic oscopy (TEM) gi es eal space images allowing a di ec in e p e a ion o he s uc u e geome y (Buschow, 2001). In TEM echnique, an elec on beam is ansmi ed h ough he sample unde obse a ion and he applied in ensi y depends on he hickness o he sample and he concen a ion o a oms in ha sample (Ebnesajjad, 2006; Hiemenz, 1997). The elec ons ha e he same ene gy and wa eleng h as hey a e accele a ed by he same ol age. TEM is no a su ace-sensi i e me hod because he elec ons pass h ough he whole sample (Bu , 2006). The C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES 79 p oduced image is a wo-dimensional p ojec ion o he en i e objec , including he su ace and he in e nal s uc u es. Di e en con as is ob ained due o he hickness and composi ion o he ma e ial and he abso p ion o elec ons in he ma e ial. TEM (Jeol JEM-1400, JEOL) images allow a mo phological analysis o he liposomes and i s complexes. Liposomes we e p epa ed as epo ed in sec ion 4.1.2, bu in his case he hyd a ion sol en used was 5% suc ose solu ion in 10 mM HEPES bu e . Suc ose allows igidi y and consequen s abili y o he esicles a e he d ying p ocess. Each sample o esicles (5 µL) was placed on coppe g ids (Fo m a /ca bon on 400 mesh – Aga ) and le o adso b o 5 minu es. The s aining was pe o med wi h 2% (w/ ) o il e ed aqueous solu ion o u anyl ace a e o 45 seconds. D ied g ids we e isualised a 80 kV and digi al Images we e collec ed a a magni ica ion o 200 000 x. 4.1.7. 4.1.7.4.1.7. 4.1.7. Complex ComplexComplex Complexa ion a iona ion a ion heo y heo y heo y heo y As in p e ious wo ks (Ba án-Be dón, 2012; Muñoz-Úbeda, 2010; Muñoz-Úbeda, 2011; Rod iguez-Pulido, 2009), we ha e used he complexa ion heo y o Nguyen and Shklo skii o s udy lipoplexes o med by ca ionic liposomes and DNA (Nguyen, 2001a; b). This phenomenological heo y desc ibes he complexa ion o a long lexible cha ged polyelec oly e o con ou leng h l, wi h opposi ely cha ged sphe ical and od-like pa icles. Acco ding o hese au ho s, he ee ene gy o he sys em o med by nega i e cha ged polyions and ca ionic liposomes F(N,x), can be exp essed as he sum o he ee ene gy o a complex o med by a single liposome and N polyions, F c (N,x), he ee ene gy o he agg ega es F a (N,x), and he ee ene gy o he emaining ee polyion in he solu ion, F p (N,x). I we pa icula ise o ou case in which polyions a e DNA molecules, he heo y p edic s ha o a gi en concen a ion o DNA (P), he s a e o he sys em can be desc ibed in e ms o 80 C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES he DNA concen a ion in he limi o new negligible small liposome concen a ion (P 0 ). To his end, a minimising calcula ion o he unc ion F(N,x) wi h espec o N and x is equi ed. As a consequence, a phase diag am can be ob ained o cha ac e ise he whole complexa ion p ocess in which lipoplexes expe ience agg ega ion (condensa ion) and disagg ega ion ( een an condensa ion) as a unc ion o he liposome-DNA a io. Namely, o a gi en concen a ion o DNA and g owing concen a ion o liposomes (S), lipoplexes expe ience agg ega ion a some c i ical concen a ion, S a , below he isoneu ali y, and emain in his agg ega ed (o condensed) s a e up o ano he concen a ion, S d , abo e he isoneu ali y. Fo concen a ions o liposomes highe han S d , clus e s o lipoplexes s a o disagg ega e as a consequence o a cha ge in e sion p ocess (Nguyen, 2001a; b). In o de o build he co esponding phase diag am, he heo y p o ides wo equa ions ha can be sol ed in e ms o he phenomenological pa ame e s P 0 and E 0 . P 0 is ela ed o he DNA concen a ion in he limi o negligible liposome concen a ions whe eas E 0 is ela ed o he ene gy gained pe complex by o ming he agg ega es (compa ed o a ee neu al isola ed liposome-DNA complex in solu ion):  !1+$% & ' (  )=*−*,-.!$' (      / % & ) (4.3) 0 !1−$% & ' (  )=*−*,-.!−$' (      / % & ) (4.4) whe e q and Q a e he o al cha ge o DNA and liposomes espec i ely,  is he he mal ene gy, C e is he elec ical capaci ance. This las pa ame e can be calcula ed as 1=4πε,ε51+κ75 i liposomes a e assumed o be sphe ical pa icles o adius a imme sed in an elec oly ic solu ion cha ac e ised by he dielec ic pe mi i i y ε,ε and a ecip ocal Debye leng h κ7. Acco dingly, C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES 81 knowing he phenomenological pa ame e s P 0 and E 0 , his se o equa ions allows he calcula ion o he wo bounda y concen a ions o liposomes S a and S d o each DNA concen a ion. Apa om his heo y, he e a e o he heo e ical and compu e simula ion s udies ha con ibu e o a be e unde s anding o DNA complexes. Fo ins ance, heo e ical analysis o he physical p ope ies o ca ionic and zwi e ionic lipid-DNA complexes based on a coa se-g ained molecula model implemen ed by Mon e Ca lo (MC) simula ions (Fa ago, 2006; Fa ago, 2007). Coa se-g ained and Molecula Dynamics Simula ions ha e been applied o s udy cha ged dend ime s in he p esence o mul i alen sal solu ion as well as hei complexa ion wi h linea polyelec oly e (Tian, 2009; 2010a; b; 2011). Also, nume ical calcula ions o he mean- ield elec os a ic ee ene gy o a zwi e ionic lipid monolaye and DNA suppo he expe imen al obse a ions whe e DNA adso bs on o a zwi e ionic lipid monolaye in he p esence o di alen ca ions (Bohinc, 2012; Mengis u, 2009). 4.1.8. 4.1.8.4.1.8. 4.1.8. Tu bidi y measu emen s Tu bidi y measu emen sTu bidi y measu emen s Tu bidi y measu emen s The u bidi y o he equilib ium phase is ela ed o he o ma ion o a non-soluble phase ha may p ecipi a e o emain s able o sho o long pe iods o ime (Espinosa-And ews, 2007). Tu bidi y measu emen s can be assessed by abso bance kine ic s udies, since agg ega ion leads o an inc ease o he a e age size o he sca e ed pa icles. Then, a spec opho ome e wo king wi h a isible wa eleng h is capable o de ec and analyse he agg ega ion kine ics o many colloidal sys ems (Peula-Ga cía, 2010). Ul a iole - isible spec oscopy (UV-Vis) e e s o abso p ion spec oscopy in he UV-Vis spec al egion. Molecules can abso b he ene gy in he o m o ul a iole o isible ligh o exci e pho ons om he g ound 82 C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES s a e o an exci ed s a e. This echnique is complemen a y o luo escence spec oscopy, p e iously desc ibed (Lakowicz, 2006). The s abili y s udies o he liposome suspensions we e pe o med by u bidi y measu emen s as a unc ion o DNA concen a ion a oom empe a u e using a UV-1700 Pha maSpec Spec ome e (Kyo o, Japan). Abso bance kine ic da a we e ob ained a 570 nm, du ing 180 seconds, wi h an in e al o 2 seconds. The liposome concen a ion was 0.5 mM and DNA was added a concen a ions anging om 0.003 o 0.150 mg/mL. The epo ed da a a e he a e age o 4 independen measu emen s. The Fuchs s abili y a io W (o s abili y ac o ) was de e mined om hese da a. This magni ude is a c i e ion o he s abili y o he colloidal sys em: W = k / k s whe e he a e cons an k desc ibes apid coagula ion and k s is he a e cons an o he slow coagula ion egime. Thus, he in e se o he s abili y a io p o ides a measu e o he e ec i eness o collisions leading o coagula ion. In o ma ion on he kine ics-agg ega ion cons an s o dime o ma ion can be di ec ly de i ed om he ini ial slopes o he abso bance s. ime cu es (dAbs/d ) (Peula-Ga cía, 2010). Acco dingly, plo ing W as a unc ion o he elec oly e concen a ion in a double-loga i hmic scale becomes use ul o es ima e he c i ical coagula ion concen a ion (CCC), which is gene ally de ined as he minimum concen a ion o elec oly e equi ed o induce coagula ion (W = 1). The CCC alue is he e o e ela ed o des abilisa ion p ocesses (i. e. low CCC means low s abili y) and i s alue has been p e iously de e mined o he case o anionic liposomes in he p esence o di alen ca ions (Ma ín-Molina, 2012). In he p esen s udy, howe e , DNA plays he ole o he elec oly e so he CCC in his case would be he minimum concen a ion o DNA equi ed o induce coagula ion o liposomes. On he o he hand, in some cases he s abili y cu es can exhibi an inc ease o W o elec oly e concen a ions la ge han he CCC. This phenomenon is usually C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES 83 called colloidal es abilisa ion and he minimum sal concen a ion a which he sys em begins o es abilise is de ined as he c i ical s abilisa ion concen a ion (CSC). This es abilisa ion phenomenon a high sal concen a ions is well known in hyd ophilic colloidal sys ems and is go e ned by hyd a ion o ces (Peula-Ga cía, 2010). In ou case, he CSC will be ede ined as he minimum DNA concen a ion a which he sys em begins o es abilise and such p ocedu e o es abilisa ion will be a ibu ed o he o ma ion o lipoplexes s abilised by he complexa ion o he DNA wi h he ca ionic liposomes. 4.1.9. 4.1.9.4.1.9. 4.1.9. Ci cula Dich oism Ci cula Dich oismCi cula Dich oism Ci cula Dich oism The DNA s uc u e when bound o he liposomes can be p edic ed by ci cula dich oism spec oscopy in he a -UV spec al egion. Ci cula dich oism is a phenomenon ha a ec s chi al molecules such as DNA and p o eins. The di e ence in abso p ion o he le (9:) and igh (9;) ci cula ly pola ised ligh de ines ci cula dich oism. CD spec opola ime e s no mally p esen he esul s as a spec um ha co ela es he wa eleng h, λ (nm), wi h he ellip ici y, θ (deg). The con e sion occu s acco ding he ollowing ule: 1=∆9=9:−9;==> ?@,AB?, (4.5) Applying he Lambe -Bee law o he CD heo y, in o de o emo e concen a ion and pa h leng h dependence, ∆9=∆C1D (4.6) whe e ∆ε is he ex inc ion coe icien (M -1 cm -1 o mol -1 dm 3 cm -1 ), C is he mola concen a ion (M) and l is he op ical pa h leng h ha c osses he sample (cm) (Rodge , 1997). The mos known a angemen o DNA molecules, disco e ed in 1953, is a igh -handed double helix o he B- o m. Howe e DNA can adop many 84 C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES o he s uc u es, o example igh -handed A- ype ypical om RNA o C- o m mos ly ound in DNA ibe s (Figu e 4.2) (Baase, 1979; Kyp , 2009; Lu, 2003; Miyaha a, 2012; Vo líčko á, 2012). Figu e 4.2. Rep esen a i e scheme o DNA A-, B- and C- o m (Lu, 2003). C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES 85 The ci cula dich oism spec a we e eco ded wi h a Jasco 720 spec opho ome e om 220 o 315 nm using qua z cu e es wi h an op ical pa h leng h o 0.5 cm. The concen a ion o DNA was kep cons an and equal o 0.05 mg/mL o cal hymus DNA (c DNA) and 0.025 mg/mL o plasmid DNA (pDNA) (pEGFP-N1, BD Biosciences Clon ech). The a e age molecula weigh conside ed o DNA was 348 g/mol (Schindle , 1997; Symie z, 2004). T is bu e (10 mM) was used as sol en . 4.1.10. 4.1.10.4.1.10. 4.1.10. In i o In i oIn i o In i o ans ec ion s udies ans ec ion s udies ans ec ion s udies ans ec ion s udies The gene ans e and ansgene exp ession a e cellula ans ec ion was moni o ed using g een luo escen p o ein (GFP) plasmid (pEGFP-N1) encoding o g een luo escen p o ein, he ea e designed by pDNA, which was ampli ied and isola ed wi h a PlasmidP ep Midi Flow ki (GE Heal hca e). The concen a ion and pu i y o pDNA we e de e mined using a NanoD op 1000 (The mo Scien i ic) appa a us. Two cell cul u e lines we e used in his s udy, SH-SY5Y and HeLa. SH-SY5Y cell line was g own in Minimum Essen ial Medium (MEM) (Lonza) and Ham’s F-12 (Lonza) (1:1), supplemen ed wi h 10% ( / ) hea inac i a ed e al bo ine se um (FBS – Gibco), 2 mM L- Glu amine (Lonza), 100U Penicillin/S ep omycin (Gibco) and 1% MEM non- essen ial amino acid solu ion (Sigma). HeLa cell line was g own in Dulbecco’s Modi ied Eagle’s Medium (Lonza) supplemen ed wi h 10% FBS and 100U Penicillin/S ep omycin. Cells we e cul u ed in a humidi ied 5% CO 2 a mosphe e a 37°C. Fo ans ec ion s udies, cells we e seeded a app oxima ely 70% con luence in 6-well issue cul u e pla es, 24 h be o e he expe imen s. Be o e ans ec ion, g ow h medium was emo ed and cells we e washed wice wi h PBS, pH 7.4. Cells we e hen incuba ed wi h 200 µL o lipoplexes p epa ed wi h liposomes and pDNA in OPTI-MEM (Gibco). The inal 86 C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES concen a ion o pDNA was 8 µg/well. The nega i e con ol co esponded o cells ea ed wi h liposomes wi hou pDNA. A e 5 h, he medium was eplaced by esh g ow h medium and ans ec ion was assessed a e 48 h incuba ion ime by low cy ome y (FACS Can o II, BD Biosciences) and CLSM. 4.1.10.1. 4.1.10.1.4.1.10.1. 4.1.10.1. Pulse cy opho ome y Pulse cy opho ome yPulse cy opho ome y Pulse cy opho ome y Pulse cy opho ome y, usually en i led low cy ome y o FACS (Fluo escence-ac i a ed cell so e ) is a biophysical echnique used o coun cells h ough de ec ion o a bioma ke inside he cell s uc u e. The de ec ion is pe o med by suspending he cells in a luid ha passes h ough a lase capable o de ec ing he bioma ke s. This echnique is use ul because i can analyse mul iple pa ame e s o indi idual cells wi hin he e ogeneous popula ions (Gi an, 2001; Shapi o, 2003). Twen y housand e en s we e measu ed o each sample. Expe imen al da a we e s a is ically analysed applying one-way analysis o a iance (ANOVA) ollowing Tukey es app oach. 4.1.10.2. 4.1.10.2.4.1.10.2. 4.1.10.2. Con ocal Lase Scanning Mic oscopy Con ocal Lase Scanning Mic oscopyCon ocal Lase Scanning Mic oscopy Con ocal Lase Scanning Mic oscopy Mic oscopy images o ans ec ed cells we e ob ained using a con ocal lase scanning mic oscope (CLSM), Leica SP2 AOBS SE (Leica Mic osys ems). Con ocal imaging is an op ical mic oscopy ool ha p o ides a high esolu ion image in a h ee dimensional o m wi hou des uc ion. This echnique has he abili y o acqui e in- ocus images om selec ed dep hs (op ical sec ioning) and ebuil a h ee-dimensional image h ough compu e echnology. This p ocess was coupled wi h luo escence mic oscopy, allowing he isualisa ion o specimens ha emi colou om luo escen ma ke s when exposed o ligh (Pawley, 2006). C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES 87 4.2. 4.2.4.2. 4.2. Resul s and Discussion Resul s and DiscussionResul s and Discussion Resul s and Discussion 4.2.1. 4.2.1.4.2.1. 4.2.1. Elec Elec Elec Elec ic p ope ies ic p ope ies ic p ope ies ic p ope ies and diame e and diame e and diame e and diame e o o o o he complexes he complexes he complexes he complexes The elec ic p ope ies o he lipoplexes we e i s ly s udied by e alua ing hei elec opho e ic mobili y ( µ e ) as a unc ion o he L/D a io. In his way, changes in he elec okine ic beha iou o he sys ems will be uni ocally ela ed o he p esence o he luo ina ed lipid F7-CHOL (hep a luo ocholes e ol) in one o he sys ems. The expe imen al da a o µ e as a unc ion o L/D a io o DOTAP:CHOL/DNA and DOTAP:F7-CHOL/DNA sys ems i o a sigmoidal cu e ha di ides he L/D axis in h ee di e en ze a po en ial egions (Figu e 4.3.A and Figu e 4.4.A): i) a egion whe e lipoplexes show a ne nega i e and almos cons an elec opho e ic mobili y; ii) a egion whe e he isoelec ic poin (L/D) 0 is eached and an in e sion o he elec opho e ic mobili y sign akes place and iii) a egion o ne posi i e elec opho e ic mobili y ha ends o he alue o he pu e liposomes. This beha iou was p e iously ound in wo ks wi h o he ca ionic lipoplexes (Ba án-Be dón, 2012; Muñoz-Úbeda, 2010; Muñoz-Úbeda, 2011; Rod iguez- Pulido, 2009). Examples o a sign e e sal in he elec opho e ic mobili y ha e been also epo ed o di e en ca ionic lipoplexes (Zuzzi, 2007) as well as o he colloidal sys ems such liposomes (Ma ín-Molina, 2010), la ex pa icles (Ma ín-Molina, 2003; Ma ín-Molina, 2008; Ma ín-Molina, 2009; Popa, 2010; Schneide , 2011) and DNA (Bes eman, 2007) in he p esence o mul i alen coun e ions. In all hese cases, he change o he sign in he elec opho e ic mobili y is explained in e ms o a cha ge in e sion (o o e cha ging) phenomena (Lyklema, 2009). 94 C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES da ke colou . The p esence o a mo e in ense colou a ound he bilaye indica es an inc ease on he densi y on he su ace o he esicles, con i ming ha DNA is compac ed on he su ace o he liposomes h ough s ong elec os a ic in e ac ions, which is in ag eemen wi h po en ial and luo escence s udies. The s ong in e ac ion be ween he liposome and DNA could ha e o he consequences. One is ha i could lead o a dis up ion on he liposomes, p oducing smalle complexes, no obse ed o hese sys ems, meaning ha hey a e e y s able wi h and wi hou DNA. Ano he consequence is ha when L/D is close o (L/D) 0 liposomes could use due o he ac ion o he condensed DNA (Rod iguez-Pulido, 2008; Rod iguez-Pulido, 2009). F om size measu emen s, o bo h sys ems, is obse ed his usion o complex agg ega ion by he inc ease o he lipoplex diame e wi h he app oxima ion o he isoneu ali y a io. On Figu e 4.7 is p esen ed some mic og aphs whe e Figu e 4.6. TEM mic og aphs o liposomes in he absence o DNA (A and D) and o lipoplexes a L/D<(L/D) 0 (B, C, E, F). (A-C) e e s o DOTAP:CHOL sys em and (D-F) o DOTAP:F7-CHOL. Scale ba : 100 nm. C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES 95 L/D a io is abo e he isoelec ic poin , o DOTAP-CHOL (A-C) and DOTAP:F7- CHOL (D-F) sys ems. In his case, i was no only no ice he inc ease on he densi y o he liposomes su ace, as well as a clea shell co e ing he en i e liposome. The exhibi ion o ami ica ions is consis en wi h he liposome coa ed by he DNA molecules. 4.2.4. 4.2.4.4.2.4. 4.2.4. Bounda y concen a ions o he Bounda y concen a ions o he Bounda y concen a ions o he Bounda y concen a ions o he lipoplexes lipoplexeslipoplexes lipoplexes The di e en elec okine ic egions ound expe imen ally o he lipoplexes as a unc ion o he L/D a io we e also analysed om a heo e ical poin o iew by using he complexa ion model p esen ed in sec ion 4.1.7. In pa icula , he model is applied o p edic he phase diag ams o he Figu e 4.7. De ails ex a ed om he o iginal TEM mic og aphs o lipoplexes a L/D>(L/D) 0 . (A-C) e e s o DOTAP:CHOL sys em and (D-F) o DOTAP:F7-CHOL. Scale ba : 100 nm. 96 C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES lipoplexes in his wo k, i. e., he calcula ion o he bounda y concen a ions S a (P) and S d (P) as a unc ion o he DNA concen a ion (P). To his end, he elec opho e ic da a a e used o ob ain he expe imen al alues o S a (P exp ) and S d (P exp ) o he DNA concen a ion ha was used in he expe imen s (Table 4.1). Then, hese inpu s a e used o sol e equa ions (4.3) and (4.4) in o de o de e mine he cha ac e is ic pa ame e s P 0 and E 0 o each sys em (Table 4.1). The expe imen al condi ions equi ed o he calcula ions (Table 4.1) a e de e mined as ollows: q is es ima ed assuming a DNA molecule o med Pa ame e 1 DOTAP:CHOL/DNA DOTAP:F7CHOL/DNA a (nm) 50.0 50.0 κ D (nm-1) 5.33 5.33 C e (C 2 J - 1 ) 1.17 x 10 - 13 1.17 x 10 - 13 Q ( e ) 28500 28500 q ( e ) 5400 5400 P exp (segmen s/l) 17.16 x 10 15 17.16 x 10 15 S a ( P exp ) (pa /l) 1.55 x 10 15 1.74 x 10 15 S d ( P exp ) (pa /l) 5.59 x 10 15 3.05 x 10 15 E 0 (J) 5.57 x 10 - 26 25.40 x 10 - 26 P 0 (segmen s/l) 5.60 x 10 15 2.91 x 10 15 1 a, liposome adius; κD, ecip ocal Debye leng h; Ce, liposome capaci ance; Q , liposome cha ge; q, cha ge o DNA segmen ; P, DNA concen a ion; Sa(P) , liposome concen a ion a agg ega ion; Sd(P), liposome concen a ion a disagg ega ion; E0 , DNA-liposome in e ac ion ene gy; P0, DNA concen a ion in equilib ium wi h lipoplexes. Table 4.1. Phenomenological pa ame e s P0 and E0 o each sys em and he equi ed expe imen al condi ions o hei calcula ion. C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES 97 by 2700 bp agmen s on a e age (ob ained om aga ose gel elec opho esis expe imen s (Muñoz-Úbeda, 2010; Rod iguez-Pulido, 2009)); Q is calcula ed om he su ace a ea o he liposome and he es ima ed a ea o he headg oup o he lipid. The phenomenological pa ame e E 0 is much la ge o DOTAP:F7-CHOL/DNA han o DOTAP:CHOL/DNA lipoplexes. This ea u e may be unde s ood as he s eng h o he liposome-DNA binding being s onge in he case o DOTAP:F7-CHOL/DNA sys em. On he o he hand, P 0 < P exp in bo h cases, which ag ees wi h he assump ion ha mos o he DNA molecules is o ming he complexes. Howe e , he alue o P 0 o DOTAP:F7-CHOL/DNA is almos hal o ha ob ained o he lipoplexes wi hou F7-CHOL. Al hough mo e DOTAP:F7-CHOL liposomes a e equi ed o o m he lipoplexes, mos o he DNA molecules could be implica ed in he complexa ion once he lipoplexes a e o med. The alues o P 0 a e simila as hose epo ed o o he cha ged lipoplexes (Ba án-Be dón, 2012; Muñoz-Úbeda, 2010; Rod iguez-Pulido, 2009). The alues o E 0 , on he o he hand, a e much lowe han hose calcula ed in he p esen wo k. This is due o he de ini ion o C e , which in he p e ious wo ks was de ined in he acuum ins ead o in an elec oly e solu ion. In any case, we mus be awa e ha P 0 and E 0 a e wo phenomenological pa ame e s and as he au ho s o he model s a ed, i is di icul o assign hem a eal meaning on he basis o a mic oscopic heo y (Nguyen, 2001a). Once he pa ame e s a e calcula ed, he alues o S a (P) and S d (P) can be p edic ed o any DNA concen a ion by a ying P in equa ions (4.3) and (4.4). Figu e 4.8 shows he heo e ical phase diag am o DOTAP:CHOL/DNA and DOTAP:F7-CHOL/DNA. Gi en a cons an alue o P, he phase diag am p edic s he exis ence o nega i ely cha ged s able lipoplexes o liposome concen a ions S < S a (P), wi h ee anionic polyelec oly e in excess. I S 98 C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES inc eases, lipoplexes expe ience agg ega ion and he suspension becomes uns able (g ey egion o he diag ams in Figu e 4.8). In his domain o S concen a ion, i.e., o S a (P) < S(P) < S d (P), he isoneu ali y o he lipoplexes is eached ( he elec opho e ic mobili y is null). Howe e , Figu e 4.8. Bounda y concen a ion lines, S a (P) and S d (P), o DOTAP:CHOL/DNA (up) and DOTAP:F7-CHOL/DNA (down) lipoplexes, acco ding o he agg ega ion- disagg ega ion heo y. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 5 10 15 20 25 30 S d (P) S a (P) S d (P exp ) S a (P exp ) 10 15 P (pa /l) 10 15 S (pa /l) DOTAP:F7-Chol/DNA P exp 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 5 10 15 20 25 30 Sd(P) Sa(P) Sd(Pexp) Sa(Pexp) 1015 P (pa /l) 1015 S (pa /l) DOTAP:Chol/DNA Pexp C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES 99 he heo y p edic s ha complexes a his poin a ac mo e ca ionic liposomes han hose equi ed o neu alise he nega i e cha ge (Nguyen, 2001a). As a consequence, a cha ge in e sion p ocess akes place. Finally, o S > S d (P), clus e s s a o pa ially dissol e gi ing ise o s able and ee posi i ely cha ged lipoplexes in coexis ence wi h he emaining clus e s. 4.2.5. 4.2.5.4.2.5. 4.2.5. S abili y a io S abili y a io S abili y a io S abili y a io Simila esul s we e ob ained o he s abili y a e o DOTAP:CHOL and DOTAP:F7-CHOL liposomes as a unc ion o DNA concen a ion (Figu e 4.9): he s abili y o he liposomes dec eases as he DNA concen a ion inc eases un il he sys ems become uns able. Then i he DNA concen a ion con inues o inc ease, bo h sys ems a e s abilised again. This beha iou is in gene al simila o ha ound o he elec opho e ic mobili y measu emen s in which wo asymp o ic alues o | µ e | we e eached o low and high alues o L/D a io. Consequen ly, egions in which he sys ems a e s able a e a ibu ed o he o ma ion o lipoplexes ha a e s abilised elec os a ically. In con as , egions whe e W ∼ 1 co esponds o a ange o DNA concen a ion a which he isoelec ic poin o he lipoplexes is expec ed o be eached. Fu he mo e, he egions whe e he sys ems a e s able and uns able ag ee wi h hose p edic ed by he complexa ion model in which a een an condensa ion p ocedu e is expec ed o happen. Conce ning he nume ical alues o CCC and CSC, sligh di e ences a e obse ed: hese c i ical DNA concen a ions a e, espec i ely, 0.022 mg/mL and 0.078 mg/m o DOTAP:CHOL. In he case o DOTAP:F7-CHOL, he alues o CCC and CSC a e, espec i ely, 0.026 mg/mL and 0.087 mg/mL. The obse ed small di e ences ag ee wi h hose ound in he esul s o elec opho e ic mobili y, which showed ha o he sys em wi h F7-CHOL lipids mo e liposomes a e equi ed o neu alise he cha ge o DNA. 100 C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES Acco dingly, he in e ac ion be ween DOTAP and luo ina ed choles e ol can 0.01 0.1 1 10 100 1000 10000 W DNA concen a ion (mg/mL) A 0.01 0.1 1 10 100 1000 10000 W DNA concen a ion (mg/mL) B Figu e 4.9. Fuchs s abili y a io W (s abili y ac o ) o liposomes DOTAP:CHOL (A) and DOTAP:F7-CHOL (B), as a unc ion o DNA concen a ion. Da a a e p esen ed on he loga i hmic scale. C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES 101 jus i y he di e en elec okine ic and s abili y beha iou ha was ound o his sys em. The ionic s eng h o physiological media can a ec he p ocess o DNA complexa ion, shi ing he s abili y a io, DNA-liposome condensa ion cu e and he isoelec ic poin o he lipoplexes, which will ha e a di ec implica ion on he DNA ans ec ion e iciency. In gene al, sal educes he elec ical su ace po en ial o he liposomes and he inc ease o he ionic s eng h shi s he isoelec ic poin o highe L/D a ios (Eas man, 1997; Kennedy, 2000; Pullmanno a, 2012). Howe e , he e ec o NaCl on he neu alisa ion is dependen on he sal concen a ion and on he ype o lipids p esen in he sys em (Eas man, 1997; Hi sch-Le ne , 2005). The DOTAP:CHOL sys em was he leas sensi i e o inc eases o NaCl and had be e in eg i y in se um when compa ed o o he s such as DOTAP:DOPE liposomes. The pe cen age o DNA ha was associa ed o he liposomes in 150 mM o NaCl aqueous solu ion was mo e han 90% o DOTAP/DNA cha ge a ios o 1:1 and 1:2 (Hi sch-Le ne , 2005). 4.2.6. 4.2.6.4.2.6. 4.2.6. DNA con o ma ion DNA con o ma ionDNA con o ma ion DNA con o ma ion The numbe o base pai s pe u n, he inclina ion o he base wi h espec o he helix axis, he dis ance o he bases om he helix axis, he ise pe base pai and he handedness o he helix cha ac e ise he polymo phism o he seconda y s uc u e o nucleic acids. Bo h c DNA and pDNA samples (Figu e 4.10) we e analysed by CD spec oscopy. They bo h show a nega i e peak a 246 nm, a posi i e peak a ound 273 nm wi h he ze o c ossing a ound 260 nm, which is consis en wi h he CD spec a o a B con o ma ion o a double helical DNA. In his case, he base pai s a e pe pendicula o he double helix axis, which con e s a weak chi ali y o he molecule, which is cha ac e ised by he low peak 102 C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES in ensi ies. The a ia ion in he ampli ude o he CD signal may be due o he di e en nucleo ide sequences (Kyp , 2009; Ma y, 2009). When DNA is elec os a ically bound o he lipid esicles, a dec ease in he Δε (M -1 cm -1 ) signal o he posi i e peak and an inc ease in he absolu e alue o he nega i e peak o he CD spec a a e obse ed (Figu e 4.11). Also bo h peaks e eal a ed wa eleng h shi . The binding o DNA o he esicles gene a es a con o ma ion change o a leas a ansi ion on he DNA polymo phism. The di e ences may sugges a pa ial B- o C-DNA con o ma ional ansi ion as s a ed be o e in he li e a u e o DOTAP complexes (Ma y, 2009). The C- o m CD spec um was ound o DNA condensed o packaged sys ems, especially monoca ionic lipid o mula ions (Ranjba , 2009; Simbe g, 2001). 220 240 260 280 300 -4 -2 0 2 4 ∆ε (Μ −1 cm −1 ) Wa eleng h (nm) c DNA pDNA Figu e 4.10. CD spec a o c DNA and pDNA in T is bu e (pH=7.4). C OMPACTION PROCESS OF DNA BY FLUORINATED LIPOSOMES 103 DNA is hus mo e compac ed in he p esence o he ca ie . 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Physicochemical cha ac e iza ion and pu i ica ion o ca ionic lipoplexes, Biophysical Jou nal, 77, 341. Zhu, L.; Maha o, R.I. (2010). Lipid and polyme ic ca ie -media ed nucleic acid deli e y, Expe Opinion on D ug Deli e y, 7, 1209. Zuzzi, S.; Came i, C.; Ono i, G.; Senna o, S. (2007). Liposome-induced DNA compac ion and een an condensa ion in es iga ed by dielec ic elaxa ion spec oscopy and dynamic ligh sca e ing echniques, Physical Re iew E, 76, 011925. B OLAAMPHIPHILE - BASED VECTORS 117 Chap e 5 5. 5.5. 5. Bola BolaBola Bolaa aa amphiphile mphiphilemphiphile mphiphile- -- -based basedbased based ec o s ec o s ec o s ec o s The p e ix “bola” is associa ed o he shape o an old Sou h Ame ican missile weapon ha consis s o wo balls a ached oge he by a co d. I was adop ed by he science communi y o ep esen isually a molecule wi h wo hyd ophilic head g oups linked by a hyd ophobic ca bon chain. The e a e ew molecules in na u e wi h hese p ope ies, howe e , mos bolaamphiphiles a e chemically syn hesized (Baek, 2010; Fuh hop, 1986). Bolaamphiphile molecules ha e hei physical s abili y enhanced and hey can be assembled in o dis inc s uc u es ha could be used as gene ca ie s (Jain, 2010). Bolaamphiphile molecules usually ha e wo simila head g oups, o igina ing a symme ic monolaye esicle. Howe e , some bolaamphiphiles a e cons i u ed by wo di e en head g oups, a posi i ely cha ged a one end and a neu al one a he o he end, assembling in o asymme ic monolaye s. Bo h ypes o bolaamphiphile can be used as esicles o encapsula e he ac i e p inciple (B unelle, 2009; G inbe g, 2010; Kau man, 2013). The e a e many applica ions o hese molecules and hey can be used no only has componen s o lipid esicles bu also in nanocapsules ha a e p epa ed by he laye -by-laye sel -assembly echnique. 118 B OLAAMPHIPHILE - BASED VECTORS 5.1. 5.1.5.1. 5.1. Bolaamphiphile pa icles Bolaamphiphile pa iclesBolaamphiphile pa icles Bolaamphiphile pa icles Bolaamphiphile molecules can be inco po a ed on pa icles h ough he deposi ion o laye s wi h di e en cha ges by laye -by-laye echnique (Figu e 5.1). I consis s on he use o a co e pa icle, such as silicon oxide (SiO 2 ), calcium ca bona e (CaCO 3 ) o polys y ene (PS). These co es a e used o p oduce mul i-laye ed ilms by laye -by-laye deposi ion o opposi ely cha ged polyme s (Shchukin, 2004; Zhao, 2006). A e deposi ion o se e al laye s (enough o allow he s abili y o he capsule) he co e can be dissol ed. The esul ing capsule will be smalle han he mic opa icle used in he p ocess due o he sh inking o he species in he absence o he co e. The ac i e p inciple can be ei he en apped o encapsula ed wi hin he ca ie . Figu e 5.1. Schema ic ep esen a ion o he Laye -by-Laye p ocess (Dona h, 1998). B OLAAMPHIPHILE - BASED VECTORS 119 5.1.1. 5.1.1.5.1.1. 5.1.1. Ma e ials and me hods Ma e ials and me hodsMa e ials and me hods Ma e ials and me hods 5.1.1.1. 5.1.1.1.5.1.1.1. 5.1.1.1. Chemicals ChemicalsChemicals Chemicals The bolaamphiphile s udied in his wo k, 22-hyd oxy-N,N,N- ime hyldocosan-1-aminium (BA), was syn he ized by P o . D . Bodo Dobne ’s g oup (Ins i u e o Pha macy, Biochemical Pha macy, Ma in Lu he Uni e si y o Halle-Wi enbe g, Halle an de Saale, Ge many) and is shown in Figu e 5.2. Monodispe se polys y ene pa icles (4.1 ± 0.1 µm, nega i ely cha ged) in a 10 % w/ aqueous suspension we e acqui ed om Mic opa icles GmbH and used as co es. DNA (deoxy ibonucleic acid sodium sal om cal hymus, ype I), sodium chlo ide (NaCl), PAH (Poly(allylamine hyd ochlo ide), posi i ely cha ged) and PSS (Poly(sodium 4-s y enesul ona e), nega i ely cha ged) we e pu chased om Sigma-Ald ich and used wi hou u he pu i ica ion. Rhod PE (1,2-dipalmi oyl-sn-glyce o-3-phosphoe hanolamine-N-(lissamine hodamine B sul onyl), ammonium sal , MW 1249.65), was pu chased om A an i Pola Lipids, Inc. The deionized wa e was pu i ied wi h Milli-Q appa a us wi h he speci ic esis ance o 18.2 MΩ∙cm. All solu ions, excep o DNA, we e il e ed h ough a 200 nm po ous memb ane. Chlo o o m was supplied by Me ck. Figu e 5.2. Chemical s uc u e o 22-hyd oxy-N,N,N- ime hyldocosan-1- aminium (BA). 126 B OLAAMPHIPHILE - BASED VECTORS 5.2.1.2. 5.2.1.2.5.2.1.2. 5.2.1.2. P epa a ion o Liposomes P epa a ion o LiposomesP epa a ion o Liposomes P epa a ion o Liposomes Liposomes we e p epa ed by he hin lipid ilm hyd a ion me hod (Palme ini, 2006). B ie ly, he app op ia e lipid mix u e (BA:DOTAP) a di e en mola a ios was dissol ed in chlo o o m, which was hen e apo a ed unde N 2 low. The lipid ilm was hyd a ed wi h 1 mL o 10 mM Hepes bu e pH 7.4 (ionic s eng h 0.00325 M). The suspension was o exed o 15 minu es, sonica ed o 15 min a 60°C and ex uded h ough Wha man il e s o dec easing po e sizes (up o 100 nm po e size memb anes) using a p essu e ex ude appa a us, a 60°C. The o al lipid concen a ion was 1 mM. 5.2.1.3. 5.2.1.3.5.2.1.3. 5.2.1.3. DLS and elec opho e ic mobili y DLS and elec opho e ic mobili yDLS and elec opho e ic mobili y DLS and elec opho e ic mobili y The hyd odynamic diame e (sec ion 4.1.3) and he elec opho e ic mobili y (sec ion 4.1.4), μ e , o liposomes and lipoplexes we e measu ed a oom empe a u e wi h a Mal e n Ze asize Nano se ies ins umen by, espec i ely, dynamic ligh sca e ing (DLS) and lase dopple elocime y. The esul s we e a e aged om h ee measu emen s o each sample and we e ob ained o a leas h ee independen p ocedu e o sample p epa a ion. The inal concen a ion o liposomes was 0.1 mg/mL and he concen a ion o pDNA was 0.01 mg/mL. 5.2.1.4. 5.2.1.4.5.2.1.4. 5.2.1.4. Mo phology s udies Mo phology s udiesMo phology s udies Mo phology s udies To cha ac e ise he ype o s uc u es o med by he bolaamphiphile alone, T ansmission Elec on Mic oscopy (TEM) and Con ocal Lase Scanning Mic oscopy (CLSM) we e used (desc ibed in sec ion 4.1.6 and 4.1.10.2, espec i ely). Fo he TEM (Jeol JEM-1400, JEOL) analysis, a d op o he B OLAAMPHIPHILE - BASED VECTORS 127 suspension was placed on coppe g ids (Fo m a /ca bon on 400 mesh – Aga ) and s ained wi h 2% (w/ ) u anyl ace a e. Fo CLSM imaging (Leica TCS SP5 II, Leica Mic osys ems) he BA was mixed wi h 0.2% o Rhod PE lipid ( luo escen ma ke ). 5.2.1.5. 5.2.1.5.5.2.1.5. 5.2.1.5. In i o In i oIn i o In i o ans ec ion s udies ans ec ion s udies ans ec ion s udies ans ec ion s udies The gene ans e and ansgene exp ession a e cellula ans ec ion was moni o ed using pDNA encoded wi h GFP. HEK 293 cell line was used in his s udy and was g own in Minimum Essen ial Medium (Lonza), supplemen ed wi h 10% ( / ) hea inac i a ed e al bo ine se um (FBS – Gibco), 2 mM L-Glu amine (Lonza), 100U Penicillin/S ep omycin (Gibco) and 1% MEM non-essen ial amino acid solu ion (Sigma). Cells we e cul u ed in a humidi ied 5% CO 2 a mosphe e a 37°C. Fo ans ec ion s udies, cells we e seeded a app oxima ely 70% con luence in 12-well issue cul u e pla es, 24 h be o e he expe imen s. Be o e ans ec ion, g ow h medium was emo ed and cells we e washed wice wi h PBS, pH 7.4. Liposome-pDNA complexes we e p epa ed by adding equal olumes o pDNA solu ion a 0.01 mg/mL o he liposome suspension. The lipid:pDNA a io was 10:1 (w:w), co esponding o a N:P a io o 5 o BA:DOTAP liposomes (cha ge a io be ween he ca ionic g oups (amine) o he liposome and he anionic g oups (phospha e) o he pDNA) (C ook, 1998; Jain, 2012; Pai a, 2013). DNA solu ion was added d op wise o he liposomes and he mix u e was kep unde magne ic s i ing o a leas 15 minu es. Cells we e hen incuba ed wi h 200 µL o lipoplexes p epa ed wi h liposomes and pDNA in OPTI-MEM (Gibco). The inal concen a ion o pDNA was 2 µg/well. The nega i e con ol co esponded o cells ea ed wi h liposomes wi hou pDNA. A e 5 h, he medium was eplaced by esh g ow h medium and ans ec ion was assessed a e 48 h 128 B OLAAMPHIPHILE - BASED VECTORS incuba ion ime by low cy ome y (FACS Can o II, BD Biosciences), de ailed in sec ion 4.1.10.1. Twen y housand e en s we e measu ed o each sample. Expe imen al da a was s a is ically analysed applying one-way analysis o a iance (ANOVA) ollowing Tukey es app oach. Mic oscopy images o ans ec ed cells we e ob ained using he CLSM. 5.2.2. 5.2.2.5.2.2. 5.2.2. Resul s and Discussion Resul s and DiscussionResul s and Discussion Resul s and Discussion 5.2.2.1. 5.2.2.1.5.2.2.1. 5.2.2.1. Vesicle cha ac e is Vesicle cha ac e isVesicle cha ac e is Vesicle cha ac e isa ion a iona ion a ion The p ope ies associa ed o bolaamphiphiles a e e y p omising wi h espec o deli e y sys ems (Nu aje, 2012). A emp s o ex uding samples con aining only BA we e no success ul, e en a high empe a u es (∼100°C). The size measu emen s e ealed a e y poly-dispe sed popula ion wi h mean diame e s o 5 ± 2 µm. CLSM and TEM analysis show ha BA o ms mic os uc u es, some o which p esen a c ys al shape (Figu e 5.5). C ys al s uc u es a e expec ed when he size o he wo pola g oups is no oo di e en . Fo ins ance, hey could assemble in o a bilaye , whe e he OH- g oups will be o ien ed o he inne space and he N- g oups o he ou e space (Figu e 2.2.a). Also, hey may o m a mixed o ien a ion whe e will be a OH- g oup and a N- g oup in he inne space, as isible on Figu e 2.2.b (Fuh hop, 2004). B OLAAMPHIPHILE - BASED VECTORS 129 P e ious wo ks epo ha hese amphiphiles sel -assemble in o small nanopa icles (50-100 nm) (Jain, 2012; Popo , 2010), sugges ing a o ma ion o an asymme ic monolaye , which was con i med by Langmui monolaye s udies and G azing incidence X-Ray di ac ion (Be chel, 2009; Deleu, 2011; Dolle, 2011; Hu e , 2012). Howe e hese s uc u es a e in luenced by he numbe o ca bons in he hyd ophobic pa o he molecule as well as he o ces p esen in he hyd ophilic pa . The hyd ophobic ail o he BA has no double bonds, which would allow he o ma ion o esicles wi h a monolaye o lipid, whe e he OH-end g oup would be di ec ed o he inside o he esicle and he N g oup o he ou side. Due o i s long ca bon chain, he BA could also ea ange i sel in a U-shape, also epo ed in he li e a u e o simila molecules (Meis e , 2007; Yan, 2009). In his case, a bilaye would be Figu e 5.5. Sel -assembled bolaamphiphile s uc u e obse ed by TEM ( op) and CLSM (bo om). Scale ba is, espec i ely, 3 and 10 µm. 130 B OLAAMPHIPHILE - BASED VECTORS o med and i s s uc u e could be mo e igid, gene a ing bigge pa icles ha would no espond so well o he ex usion me hod due o i s assembly. Mixing his BA wi h o he lipids a app op ia e a ios could help he o ma ion o la ge unilamella esicles (LUV), whe e he BA could be in e cala ed wi h he o he lipids. The BA was mixed wi h a well-known and commonly used ca ionic lipid, DOTAP (Zhu, 2010), a BA:DOTAP mola a ios o 1:1, 1:5 and 1:10. Fo he a io 1:1, i was no possible o ca y ou ex usion o he sample. The size o he s uc u es was smalle han ha ob ained o he BA alone (Figu e 5.6). A he mola a ios o 1:5 and 1:10, BA:DOTAP esicles we e ex uded and i was possible o ob ain la ge unilamella esicles using il e s o po e size o 100 nm. Due he simila i y o he esul s o he wo a ios, esicles p epa ed wi h BA:DOTAP a 1:5 mola a ios we e chosen o u he s udies BA 1:1 1:5 1:10 DOTAP 0 40 80 120 160 1500 3000 4500 6000 7500 Size (nm) Sys em ( a io BA:DOTAP) Figu e 5.6. Hyd odynamic diame e o esicles o BA, DOTAP and hei mix u e a di e en mola a ios. B OLAAMPHIPHILE - BASED VECTORS 131 using cell cul u es. In he li e a u e, mix u es o bolaamphiphiles wi h o he lipids, such as choles e ol (Dakwa , 2012) and DOPE (Jain, 2012) ha e been epo ed, mainly o s abilise he liposomes when hey a e complexed wi h pDNA. Since he BA does no sel -assemble in o small esicles, DOTAP was chosen due i s cha ge, which will help he o ma ion o s able nanopa icles e en when hey a e complexed wi h pDNA. Ze a po en ial o he esicles was measu ed o he o mula ions in s udy (Figu e 5.7). The complex o ma ion wi h DNA was obse ed a he a io o lipid:pDNA 10:1 (w:w) (Pai a, 2013). To assess he s abili y o he sys em, ze a po en ial measu emen s we e conduc ed wi h and wi hou pDNA. Size measu emen s o he liposomes and he lipoplexes e ealed simila alues o ha ob ained o he liposomes alone, howe e , he pDNA has an e ec on he popula ion dis ibu ion educes he polydispe si y index (PdI) o he BA BA:DOTAP 1:5 DOTAP 0 10 20 30 40 50 Ze a Po en ial (mV) Sys em Vesicle Complex Figu e 5.7. Ze a po en ial alues o he s udied o mula ions, be o e and a e complexa ion. 132 B OLAAMPHIPHILE - BASED VECTORS sample. Ze a po en ial alues we e also ob ained o he o mula ions and showed we e a ound 32 mV o all p epa a ions (Figu e 5.7). This pa ame e indica es ha all lipoplexes a e s able and posi i ely cha ged, which is a condi ion desi able o cell ans ec ions s udies (Jeso ka, 2008; Lonez, 2008). 5.2.2.2. 5.2.2.2.5.2.2.2. 5.2.2.2. Cell ans ec ion s udies Cell ans ec ion s udiesCell ans ec ion s udies Cell ans ec ion s udies The ans ec ion e iciency o lipoplexes was e alua ed using HEK 293 cell line, a human emb yonic kidney de i ed cell line. Cells we e ans ec ed wi h plasmid DNA (pDNA) encoding g een luo escen p o ein (GFP). Flow cy ome y analysis e ealed ha GFP exp ession was induced simila ly by DOTAP/pDNA and BA:DOTAP/pDNA (Figu e 5.8.A). Naked pDNA did no ans ec he cells (Figu e 5.8.B). DOTAP has been p o en o be an e icien and wide used sys em o complex, deli e and ans ec DNA in o, o example, human hema opoie ic s em cells (Ma ino, 2009) and ce ical cance cells (Fle che , 2005). Combined wi h o he lipids, like choles e ol, DOTAP has been s udied in i o and shown o be e icien in anspo ing gene ic ma e ial o cells (Eliyahu, 2007; Kim, 2007; Ramesh, 2001; Temple on, 1997). These esul s show ha he BA mixed wi h DOTAP a he mola a io o 1:5, has po en ial as ehicle o pDNA deli e y, e idencing good esul s wi h espec o he DOTAP sys em. I was obse ed o all cell ials ha BA:DOTAP sys em achie ed highe alues han DOTAP sys em. Howe e and acco ding o he s a is ical analysis, he ans ec ion alues a e no signi ican ly di e en a he 0.05 le el, indica ing ha bo h sys ems ha e a simila abili y o ans ec cells. The cy ome y echnique and con ocal analysis showed ha DOTAP lipoplexes cause mo e cellula dead han BA:DOTAP lipoplexes, indica ing ha his sys em is less cy o oxic. B OLAAMPHIPHILE - BASED VECTORS 133 5.2.3. 5.2.3.5.2.3. 5.2.3. Conclusions ConclusionsConclusions Conclusions We p opose esicles ha combine a bolaamphiphile wi h ca ionic lipids o o m s able and posi i ely cha ged liposomes capable o compac ing e icien ly gene ic ma e ial by elec os a ic in e ac ions. Compa ing he ca ie D O T A P B A :D O T A P 1 :5 0 5 1 0 1 5 2 0 2 5 3 0 3 5 % T ans ec ed cells A Figu e 5.8. T ans ec ion o HEK cell line wi h g een luo escen p o ein (GFP) plasmid (pDNA) a 10:1 L/D a io: (A) cy ome y analysis o ans ec ion le els o DOTAP and BA:DOTAP 1:5 sys ems. Resul s a e exp essed as mean ± S.D. (n=3). (B) Illus a i e con ocal images o GFP exp ession when ans ec ed wi h naked pDNA (B1), DOTAP (B2) and BA:DOTAP 1:5 (B3). The scale ba is 50 µm. 134 B OLAAMPHIPHILE - BASED VECTORS made o BA:DOTAP 1:5 wi h DOTAP esicles, hey p esen simila p ope ies wi h espec o size and ze a po en ial alues. The ans ec ion s udies show ha he bolaamphiphile mixed sys em has a highe ans ec ion po en ial when compa ed o DOTAP esicles. 5.3. 5.3.5.3. 5.3. R RR Re e en e e ene e en e e ences cesces ces Baek, K.; Kim, Y.; Kim, H.; Yoon, M.; Hwang, I.; Ko, Y.H.; Kim, K. (2010). 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