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).
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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
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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
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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).
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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.

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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.
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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
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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
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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
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wi h Fluo ina ed Glyce ophosphoe hanolamine Helpe Lipids,
Bioconjuga e Chemis y, 12, 949.
Gauche on, J.; San aella, C.; Vie ling, P. (2001b). Imp o ed in i o gene
ans e media ed by luo ina ed lipoplexes in he p esence o a bile
sal su ac an , The Jou nal o Gene Medicine, 3, 338.
Gauche on, J.; San aella, C.; Vie ling, P. (2002). T ans ec ion wi h luo ina ed
lipoplexes based on luo ina ed analogues o DOTMA, DMRIE and
DPPES, Biochimica e Biophysica Ac a (BBA) - Biomemb anes, 1564,
349.
Geusens, B.; S obbe, T.; B acke, S.; Dynood , P.; Sande s, N.; Gele, M.V.;
Lambe , J. (2011). Lipid-media ed gene deli e y o he skin,
Eu opean Jou nal o Pha maceu ical Sciences, 43, 199.
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.
G inbe g, S.; Kipnis, N.; Linde , C.; Kolo , V.; Heldman, E. (2010). Asymme ic
bolaamphiphiles om e nonia oil designed o d ug deli e y,
Eu opean Jou nal o Lipid Science and Technology, 112, 137.
Gu, Q.; Zou, A.; Yuan, C.; Guo, R. (2003). E ec s o a bolaamphiphile on he
s uc u e o phospha idylcholine liposomes, Jou nal o Colloid and
In e ace Science, 266, 442.
Gu, Y.; Zhang, J.; Guo, L.; Cui, S.; Li, X.; Ding, D.; Kim, J.-M.; Ho, S.-H.; Hahn, W.;
Kim, S. (2011). A phase I clinical s udy o naked DNA exp essing wo
iso o ms o hepa ocy e g ow h ac o o ea pa ien s wi h c i ical
limb ischemia, The Jou nal o Gene Medicine, 13, 602.
Hal e , M.; Noga a, Y.; Dannenbe ge , O.; Sasaki, T.; Vogel, V. (2004).
Enginee ed Lipids Tha C oss-Link he Inne and Ou e Lea le s o
Lipid Bilaye s, Langmui , 20, 2416.

N
ANO
-
SYSTEMS FOR GENE DELIVERY
31
Han, J.; Il Yeom, Y. (2000). Speci ic gene ans e media ed by galac osyla ed
poly-l-lysine in o hepa oma cells, In e na ional Jou nal o
Pha maceu ics, 202, 151.
He weije , H.; Wol , J.A. (2003). P og ess and p ospec s: naked DNA gene
ans e and he apy, Gene The apy, 10, 453.
Honda, M.; Asai, T.; Oku, N.; A aki, Y.; Tanaka, M.; Ebiha a, N. (2013).
Liposomes and nano echnology in d ug de elopmen : Focus on
ocula a ge s, In e na ional Jou nal o Nanomedicine, 8, 495.
Huang, H.-C.; Ba ua, S.; Sha ma, G.; Dey, S.K.; Rege, K. (2011). Ino ganic
nanopa icles o cance imaging and he apy, Jou nal o Con olled
Release, 155, 344.
Huh, N.W.; Po e , N.A.; McIn osh, T.J.; Simon, S.A. (1996). The in e ac ion o
polyphenols wi h bilaye s: Condi ions o inc easing bilaye adhesion,
Biophysical Jou nal, 71, 3261.
Hui, S.W.; Langne , M.; Zhao, Y.L.; Ross, P.; Hu ley, E.; Chan, K. (1996). The
ole o helpe lipids in ca ionic liposome-media ed gene ans e ,
Biophysical Jou nal, 71, 590.
Hu e , T.; Linde , C.; Heldman, E.; G inbe g, S. (2012). In e acial and sel -
assembly p ope ies o bolaamphiphilic compounds de i ed om a
mul i unc ional oil, Jou nal o Colloid and In e ace Science, 365, 53.
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.
Jain, N.; Goldschmid , V.; Oncul, S.; A n z, Y.; Dupo ail, G.; Mély, Y.;
Klymchenko, A.S. (2012). Lac ose-o ni hine bolaamphiphiles o
e icien gene deli e y in i o, In e na ional Jou nal o
Pha maceu ics, 423, 392.
32 N
ANO
-
SYSTEMS FOR GENE DELIVERY
Janciauskai e, U.; Raku y e, V.; Miskinis, J.; Makuska, R. (2008). Syn hesis and
p ope ies o chi osan-N-dex an g a copolyme s, Reac i e and
Func ional Polyme s, 68, 787.
Jayakuma , R.; Chennazhi, K.P.; Muzza elli, R.A.A.; Tamu a, H.; Nai , S.V.;
Sel amu ugan, N. (2010a). Chi osan conjuga ed DNA nanopa icles in
gene he apy, Ca bohyd a e Polyme s, 79, 1.
Jayakuma , R.; P abaha an, M.; Nai , S.V.; Toku a, S.; Tamu a, H.;
Sel amu ugan, N. (2010b). No el ca boxyme hyl de i a i es o chi in
and chi osan ma e ials and hei biomedical applica ions, P og ess in
Ma e ials Science, 55, 675.
Jean, M.; Alameh, M.; Buschmann, M.D.; Me zouki, A. (2011). E ec i e and
sa e gene-based deli e y o GLP-1 using chi osan/plasmid-DNA
he apeu ic nanocomplexes in an animal model o ype 2 diabe es,
Gene The apy, 18, 807.
Jeong, J.H.; Pa k, T.G. (2002). Poly(l-lysine)-g-poly(d,l-lac ic-co-glycolic acid)
micelles o low cy o oxic biodeg adable gene deli e y ca ie s,
Jou nal o Con olled Release, 82, 159.
Jeso ka, A.; O wa , O. (2008). Liposomes: Technologies and Analy ical
Applica ions, Annual Re iew o Analy ical Chemis y, 1, 801.
Jewell, C.M.; Lynn, D.M. (2008). Su ace-media ed deli e y o DNA: Ca ionic
polyme s ake cha ge, Cu en Opinion in Colloid & In e ace Science,
13, 395.
Kami, D.; Takeda, S.; I aku a, Y.; Gojo, S.; Wa anabe, M.; Toyoda, M. (2011).
Applica ion o Magne ic Nanopa icles o Gene Deli e y,
In e na ional Jou nal o Molecula Sciences, 12, 3705.
Kamimu a, K.; Suda, T.; Guisheng, Z.; Dexi, L. (2011). Ad ances in Gene
Deli e y Sys ems, Pha maceu ical Medicine - New Zealand, 25, 293.
N
ANO
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SYSTEMS FOR GENE DELIVERY
33
Kaneko, Y.; Ma suda, S.I.; Kadokawa, J.I. (2007). Chemoenzyma ic syn hesis o
amylose-g a ed chi in and chi osan, Biomac omolecules, 8, 3959.
Kau man, Y.; G inbe g, S.; Linde , C.; Heldman, E.; Gil on, J.; F ege , V. (2013).
Fusion o bolaamphiphile micelles: A me hod o p epa e s able
suppo ed biomime ic memb anes, Langmui , 29, 1152.
Kay, M.A.; Glo ioso, J.C.; Naldini, L. (2001). Vi al ec o s o gene he apy: he
a o u ning in ec ious agen s in o ehicles o he apeu ics, Na u e
Medicine, 7, 33.
Kay, M.A. (2011). S a e-o - he-a gene-based he apies: The oad ahead,
Na u e Re iews Gene ics, 12, 316.
Kiang, T.; Wen, J.; Lim, H.W.; Leong, K.W.K.W. (2004). The e ec o he deg ee
o chi osan deace yla ion on he e iciency o gene ans ec ion,
Bioma e ials, 25, 5293.
Kiem, H.-P.; Je ome, Kei h R.; Deeks, S e en G.; McCune, Joseph M. (2012).
Hema opoie ic-S em-Cell-Based Gene The apy o HIV Disease, Cell
S em Cell, 10, 137.
Kim, S.U.; de Vellis, J. (2009). S em cell-based cell he apy in neu ological
diseases: A e iew, Jou nal o Neu oscience Resea ch, 87, 2183.
Kim, T.-H.; Jiang, H.-L.; Je e, D.; Pa k, I.-K.; Cho, M.-H.; Nah, J.-W.; Choi, Y.-J.;
Akaike, T.; Cho, C.-S. (2007). Chemical modi ica ion o chi osan as a
gene ca ie in i o and in i o, P og ess in Polyme Science, 32, 726.
Kissa, E.; Fluo ina ed Su ac an s: Syn hesis, P ope ies, Applica ions, Vol. 50,
Ma cel Dekke , Uni ed S a es o Ame ica, 1994.
Klein, E.; Ciobanu, M.; Klein, J.; MacHi, V.; Lebo gne, C.; Vandamme, T.; F isch,
B.; Pons, F.; Kichle , A.; Zube , G.; Lebeau, L. (2010). "HFP" luo ina ed
ca ionic lipids o enhanced lipoplex s abili y and gene deli e y,
Bioconjuga e Chemis y, 21, 360.
34 N
ANO
-
SYSTEMS FOR GENE DELIVERY
K a , M.P.; Riess, J.G. (1998). Highly luo ina ed amphiphiles and colloidal
sys ems, and hei applica ions in he biomedical ield. A
con ibu ion, Biochimie, 80, 489.
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.
K a , M.P.; Goldmann, M. (2003). Monolaye s made om luo ina ed
amphiphiles, Cu en Opinion in Colloid & In e ace Science, 8, 243.
Laouini, A.; Cha cosse , C.; Fessi, H.; Holdich, R.G.; Vladisa lje ic, G.T. (2013).
P epa a ion o liposomes: a no el applica ion o mic oenginee ed
memb anes - in es iga ion o he p ocess pa ame e s and applica ion
o he encapsula ion o i amin E, RSC Ad ances, 3, 4985.
Leal, C.; Moni i, E.; Pegado, L.; Wenne s om, H. (2007). Elec os a ic
a ac ion be ween DNA and a ca ionic su ac an agg ega e. The
sc eening e ec o sal , Jou nal o Physical Chemis y B, 111, 5999.
Leese, P.T.; No eck, R.J.; Sho , J.S.; Woods, C.M.; Flaim, K.E.; Keipe , P.E.
(2000). Randomized sa e y s udies o in a enous pe lub on
emulsion. I. E ec s on coagula ion unc ion in heal hy olun ee s,
Anes hesia and Analgesia, 91, 804.
Le en is, R.; Sil ius, J.R. (1990). In e ac ions o mammalian cells wi h lipid
dispe sions con aining no el me abolizable ca ionic amphiphiles,
Biochimica e Biophysica Ac a (BBA) - Biomemb anes, 1023, 124.
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.
Li, D.; Li, P.; Li, G.; Wang, J.; Wang, E. (2009). The e ec o nocodazole on he
ans ec ion e iciency o lipid-bilaye coa ed gold nanopa icles,
Bioma e ials, 30, 1382.
N
ANO
-
SYSTEMS FOR GENE DELIVERY
35
Li, D.; Li, G.; Li, P.; Zhang, L.; Liu, Z.; Wang, J.; Wang, E. (2010). The
enhancemen o ans ec ion e iciency o ca ionic liposomes by
didodecyldime hylammonium b omide coa ed gold nanopa icles,
Bioma e ials, 31, 1850.
Liu, Z.; Jiao, Y.; Wang, Y.; Zhou, C.; Zhang, Z. (2008). Polysaccha ides-based
nanopa icles as d ug deli e y sys ems, Ad anced D ug Deli e y
Re iews, 60, 1650.
Lonez, C.; Vandenb anden, M.; Ruysschae , J.-M. (2008). Ca ionic liposomal
lipids: F om gene ca ie s o cell signaling, P og ess in Lipid Resea ch,
47, 340.
Luo, D.; Sal zman, W.M. (2000). Syn he ic DNA deli e y sys ems, Na u e
Bio echnology, 18, 33.
Man edsson, F.P.; Mandel, R.J. (2010). De elopmen o gene he apy o
neu ological diso de s, Disco e y medicine, 9, 204.
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.
Mao, S.; Sun, W.; Kissel, T. (2010). Chi osan-based o mula ions o deli e y o
DNA and siRNA, Ad anced D ug Deli e y Re iews, 62, 12.
Maso i, A.; O aggi, G. (2009). Chi osan Mic o- and Nanosphe es: Fab ica ion
and Applica ions o D ug and DNA Deli e y, Mini-Re iews in
Medicinal Chemis y, 9, 463.
Ma hew, M.E.; Mohan, J.C.; Manzoo , K.; Nai , S.V.; Tamu a, H.; Jayakuma , R.
(2010). Fola e conjuga ed ca boxyme hyl chi osan-manganese doped
zinc sulphide nanopa icles o a ge ed d ug deli e y and imaging o
cance cells, Ca bohyd a e Polyme s, 80, 442.

36 N
ANO
-
SYSTEMS FOR GENE DELIVERY
Meis e , A.; Weygand, M.J.; B ezesinski, G.; Ke h, A.; D esche , S.; Dobne , B.;
Blume, A. (2007). E idence o a Re e se U-Shaped Con o ma ion o
Single-Chain Bolaamphiphiles a he Ai −Wa e In e ace, Langmui ,
23, 6063.
Me dan, T.; Kopecek, J.; Kissel, T. (2002). P ospec s o ca ionic polyme s in
gene and oligonucleo ide he apy agains cance , Ad anced D ug
Deli e y Re iews, 54, 715.
Miguel, M.G.; Pais, A.; Dias, R.S.; Leal, C.; Rosa, M.; Lindman, B. (2003). DNA-
ca ionic amphiphile in e ac ions, Colloids and Su aces A:
Physicochemical and Enginee ing Aspec s, 228, 43.
Min ze , M.A.; Simanek, E.E. (2009). Non i al Vec o s o Gene Deli e y,
Chemical Re iews, 109, 259.
Mi omo, K.; G iesenbach, U.; Inoue, M.; Some on, L.; Meng, C.; Akiba, E.;
Taba a, T.; Ueda, Y.; F ankel, G.M.; Fa ley, R.; Singh, C.; Chan, M.;
Munkonge, F.; B um, A.; Xena iou, S.; Escude o-Ga cia, S.; Hasegawa,
M.; Al on, E.W. (2010). Towa d gene he apy o cys ic ib osis using a
len i i us pseudo yped wi h Sendai i us en elopes, Molecula
The apy, 18, 1173.
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.
Mo ishi a, R.; Makino, H.; Aoki, M.; Hashiya, N.; Yamasaki, K.; Azuma, J.;
Taniyama, Y.; Sawa, Y.; Kaneda, Y.; Ogiha a, T. (2011). Phase I/IIa
Clinical T ial o The apeu ic Angiogenesis Using Hepa ocy e G ow h
Fac o Gene T ans e o T ea C i ical Limb Ischemia, A e ioscle osis,
Th ombosis, and Vascula Biology, 31, 713.
N
ANO
-
SYSTEMS FOR GENE DELIVERY
37
Mose , C.; Amacke , M.; Zu b iggen, R. (2011). In luenza i osomes as a
accine adju an and ca ie sys em, Expe Re iew o Vaccines, 10,
437.
Moss, R.A.; Fuji a, T.; Okumu a, Y. (1991). Dynamics o a bolaamphiphilic lipid
in a bilaye liposome, Langmui , 7, 2415.
Muñoz-Úbeda, M.; Rod íguez-Pulido, A.; Nogales, A.; Ma ín-Molina, A.;
Aica , E.; Junque a, E. (2010). E ec o lipid composi ion on he
s uc u e and heo e ical phase diag ams o DC-Chol/DOPE-DNA
lipoplexes, Biomac omolecules, 11, 3332.
Naahidi, S.; Ja a i, M.; Edala , F.; Raymond, K.; Khademhosseini, A.; Chen, P.
(2013). Biocompa ibili y o enginee ed nanopa icles o d ug
deli e y, Jou nal o Con olled Release, 166, 182.
Nayak, S.; He zog, R.W. (2010). P og ess and p ospec s: Immune esponses o
i al ec o s, Gene The apy, 17, 295.
Nguyen, D.N.; G een, J.J.; Chan, J.M.; Longe , R.; Ande son, D.G. (2009).
Polyme ic Ma e ials o Gene Deli e y and DNA Vaccina ion,
Ad anced Ma e ials, 21, 847.
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
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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). Nanopa icles: a boon o d ug
deli e y, he apeu ics, diagnos ics and imaging, Nanomedicine:
Nano echnology, Biology and Medicine, 8, 147.
Pa el, T.; Zhou, J.; Piepmeie , J.M.; Sal zman, W.M. (2012). Polyme ic
nanopa icles o d ug deli e y o he cen al ne ous sys em,
Ad anced D ug Deli e y Re iews, 64, 701.
Pa il, S.; Rhodes, D.; Bu gess, D. (2004). Anionic liposomal deli e y sys em o
DNA ans ec ion, The AAPS Jou nal, 6, 13.
Pa il, S.; Rhodes, D.; Bu gess, D. (2005a). DNA-based he apeu ics and DNA
deli e y sys ems: A comp ehensi e e iew, The AAPS Jou nal, 7, E61.
Pa il, S.D.; Rhodes, D.G.; Bu gess, D.J. (2005b). Biophysical cha ac e iza ion o
anionic lipoplexes, Biochimica e Biophysica Ac a (BBA) -
Biomemb anes, 1711, 1.
Ped oso de Lima, M.C.; Ne es, S.; Filipe, A.; Düzgüneş, N.; Simões, S. (2003).
Ca ionic liposomes o gene deli e y: om biophysics o biological
applica ions, Cu en Medicinal Chemis y, 10, 1221.
Pe e sen, H.; Fechne , P.M.; Fische , D.; Kissel, T. (2002). 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. Re e ences
Re e encesRe e ences
Re e ences
And ee a, T.D.; Pe o , J.G.; B ezesinski, G.; Moehwald, H. (2008). S uc u e o
he Langmui monolaye s wi h luo ina ed e hyl amide and e hyl
es e pola heads c ea ing dipole po en ials o opposi e sign,
Langmui , 24, 8001.
Banyay, M.; Sa ka , M.; G äslund, A. (2003). A lib a y o IR bands o nucleic
acids in solu ion, Biophysical Chemis y, 104, 477.
Be ne , M.K.; Zisman, W.A. (1963). Beha io o Monolaye s o P og essi ely
Fluo ina ed Fa y Acids Abso bed on Wa e , Jou nal o Physical
Chemis y, 67, 1534.
Blume, A. (1996). P ope ies o lipid esicles: FT-IR spec oscopy and
luo escence p obe s udies, Cu en Opinion in Colloid & In e ace
Science, 1, 64.
Bo di, F.; Came i, C.; Senna o, S.; Paoli, B.; Ma ianecci, C. (2006). Cha ge
eno maliza ion in plana and sphe ical cha ged lipidic aqueous
in e aces, Jou nal o Physical Chemis y B, 110, 4808.
B ezesinski, G.; Möhwald, H. (2003). Langmui monolaye s o s udy
in e ac ions a model memb ane su aces, Ad ances in Colloid and
In e ace Science, 100-102, 563.
Buschow, K.H.J.C., Robe W.; Flemings, Me on C.; Ilschne , Be nha d;
K ame , Edwa d J.; Mahajan, Subhash; Encyclopedia o Ma e ials -
I
NTERACTION OF
DNA
WITH LIPID MONOLAYERS
65
Science and Technology, Volume 1-11, 1s Edi ion, Else ie , The
Ne he lands, 2001.
Bu , H.-J.; G a , K.; Physics and chemis y o in e aces, Wiley-VCH, Ge many,
2006.
Daly, T.A.; Wang, M.H.; Regen, S.L. (2011). The O igin o Choles e ol's
Condensing E ec , Langmui , 27, 2159.
Demel, R.A.; Vandeene.Ll; Pe hica, B.A. (1967). Monolaye In e ac ions o
Phospholipids and Choles e ol, Biochimica E Biophysica Ac a, 135,
11.
Di ich, M.; Bo che , M.; Oli ei a, J.S.L.; Dobne , B.; Mohwald, H.;
B ezesinski, G. (2011). Physical-chemical cha ac e iza ion o no el
ca ionic ans ec ion lipids and he binding o model DNA a he ai -
wa e in e ace, So Ma e , 7, 10162.
Dopico, A.M.; Me hods in Memb ane Lipids, Volume 400, 16 h Edi ion,
Humana P ess, Uni ed S a es o Ame ica, 2007.
Faine man, V.B.; Lucassen-Reynde s, E.H.; Mille , R. (1998). Adso p ion o
su ac an s and p o eins a luid in e aces, Colloids and Su aces A:
Physicochemical and Enginee ing Aspec s, 143, 141.
Fox, H.W. (1957). Fo ce-A ea and Po en ial-A ea Rela ions o Monolaye s o
Te minally Fluo ina ed Oc adecylamine and Oc adecanoic Acid,
Jou nal o Physical Chemis y, 61, 1058.
F ei e, M.G.; Ca alho, P.J.; Fe nandes, A.M.; Ma ucho, I.M.; Queimada, A.J.;
Cou inho, J.A.P. (2007). Su ace ensions o imidazolium based ionic
liquids: Anion, ca ion, empe a u e and wa e e ec , Jou nal o
Colloid and In e ace Science, 314, 621.
G omelski, S.; B ezesinski, G. (2004). Adso p ion o DNA o zwi e ionic DMPE
monolaye s media ed by magnesium ions, Physical Chemis y
Chemical Physics, 6, 5551.
66 I
NTERACTION OF
DNA
WITH LIPID MONOLAYERS
G omelski, S.; B ezesinski, G. (2006). DNA condensa ion and in e ac ion wi h
zwi e ionic phospholipids media ed by di alen ca ions, Langmui ,
22, 6293.
Halda , S.; Kanapa hi, R.K.; Saman a, A.; Cha opadhyay, A. (2012).
Di e en ial E ec o Choles e ol and I s Biosyn he ic P ecu so s on
Memb ane Dipole Po en ial, Biophysical Jou nal, 102, 1561.
Henon, S.; Meunie , J. (1991). Mic oscope a he B ews e angle: Di ec
obse a ion o i s -o de phase ansi ions in monolaye s, Re iew o
Scien i ic Ins umen s, 62, 936.
Kae che , T.; Hönig, D.; Möbius, D. (1993). B ews e angle mic oscopy,
In e na ional Oph halmology, 17, 341.
Kau man, J.M.; Wes e man, P.W.; Ca ey, M.C. (2000). Fluo ocholes e ols, in
con as o hyd oxycholes e ols, exhibi in e acial p ope ies simila
o choles e ol, Jou nal o Lipid Resea ch, 41, 991.
Lea hes, J.B. (1925). Role o a s in i al phenomena., Lance , 1, 853.
Lewis, R.N.A.H.; McElhaney, R.N. (1998). The s uc u e and o ganiza ion o
phospholipid bilaye s as e ealed by in a ed spec oscopy, Chemis y
and Physics o Lipids, 96, 9.
Macphail, R.A.; S auss, H.L.; Snyde , R.G.; Ellige , C.A. (1984). C-H S e ching
Modes and he S uc u e o No mal-Alkyl Chains .2. Long, All-T ans
Chains, Jou nal o Physical Chemis y, 88, 334.
Mal se a, E.; Model memb ane in e ac ions wi h ions and pep ides a he
ai /wa e in e ace, Ph.D, Uni e si ä Po sdam, 2005.
McIn osh, T.J.; Simon, S.A.; Vie ling, P.; San aella, C.; Ra ily, V. (1996).
S uc u e and in e ac i e p ope ies o highly luo ina ed
phospholipid bilaye s, Biophysical Jou nal, 71, 1853.
Melze , V.; Weidemann, G.; Vollha d , D.; B ezesinski, G.; Wagne , R.; S u h,
B.; Möhwald, H. (1997). B ews e Angle Mic oscopy and X- ay GID
I
NTERACTION OF
DNA
WITH LIPID MONOLAYERS
67
S udies o Mo phology and C ys al S uc u e in Monolaye s o N-
Te adecyl-γ,δ-dihyd oxypen anoic Acid Amide, The Jou nal o
Physical Chemis y B, 101, 4752.
Mendelsohn, R.; Mao, G.; Flach, C.R. (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).
Mo phological and S uc u al Cha ac e is ics o Monoglyce ide
Monolaye s a he Ai −Wa e In e ace Obse ed by B ews e Angle
Mic oscopy, Langmui , 15, 2484.
Shapo alo , V.L.; B ezesinski, G. (2006). B eakdown o he Gouy−Chapman
Model o Highly Cha ged Langmui Monolaye s:  Coun e ion Size
E ec , The Jou nal o Physical Chemis y B, 110, 10032.
Shapo alo , V.L.; Di ich, M.; Kono alo , O.V.; B ezesmski, G. (2010). Use o
To al Re lec ion X- ay Fluo escence (TRXF) o he Quan i ica ion o

68 I
NTERACTION OF
DNA
WITH LIPID MONOLAYERS
DNA Binding o Lipid Monolaye s a he Ai -Wa e In e ace,
Langmui , 26, 14766.
Shiba a, O.; Yamamo o, S.K.; Lee, S.; Sugiha a, G. (1996). Mixed monolaye
p ope ies o e adecanoic acid wi h n-pe luo oca boxylic acids wi h
10, 12, 14, 16, and 18 ca bon a oms, Jou nal o Colloid and In e ace
Science, 184, 201.
Snyde , R.G.; S auss, H.L.; Ellige , C.A. (1982). C-H S e ching Modes and he
S uc u e o No mal-Alkyl Chains .1. Long, Diso de ed Chains, Jou nal
o Physical Chemis y, 86, 5145.
S ock on, G.W.; Smi h, I.C.P. (1976). Deu e ium Nuclea Magne ic-Resonance
S udy o Condensing E ec o Choles e ol on Egg Phospha idylcholine
Bilaye Memb anes .1. Pe deu e a ed Fa y-Acid P obes, Chemis y
and Physics o Lipids, 17, 251.
Symie z, C.; Schneide , M.; B ezesinski, G.; Möhwald, H. (2004). DNA
alignmen a ca ionic lipid monolaye s a he ai /wa e in e ace,
Mac omolecules, 37, 3865.
T uzzolillo, D.; Bo di, F.; Came i, C.; Senna o, S. (2008). Phenomenological
su ace cha ac e iza ion o ca ionic-lipid monolaye s in he p esence
o opposi ely cha ged polyions, Colloids and Su aces a-
Physicochemical and Enginee ing Aspec s, 319, 51.
Vogel, V.; Mobius, D. (1988). Local Su ace-Po en ials and Elec ic-Dipole
Momen s o Lipid Monolaye s - Con ibu ions o he Wa e Lipid and
he Lipid Ai In e aces, Jou nal o Colloid and In e ace Science, 126,
408.
Vollha d , D.; Faine man, V.B. (2006). 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 . The use o
he luo ina ed helpe lipid (F7-CHOL) has no impac on he con o ma ional
220 240 260 280 300
-3
-2
-1
0
1
2
3
∆ε (Μ
−1
cm
−1
)
Wa eleng h (nm)
c DNA
DOTAP:CHOL/c DNA
DOTAP:F7-CHOL/c DNA
220 240 260 280 300
-6
-4
-2
0
2
4
6
Wa eleng h (nm)
∆ε (Μ
−1
cm
−1
)
pDNA
DOTAP:CHOL/pDNA
DOTAP:F7-CHOL/pDNA
Figu e 4.11. CD spec a o c DNA (up) and pDNA (down) o DNA alone and
complexes o DNA wi h DOTAP:CHOL and DOTAP:F7-CHOL a he L/D a io o
10:1.
110 C
OMPACTION PROCESS OF
DNA
BY FLUORINATED LIPOSOMES
Goncal es, E.; Debs, R.J.; Hea h, T.D. (2004). The e ec o liposome size on he
inal lipid/DNA a io o ca ionic lipoplexes, Biophysical Jou nal, 86,
1554.
Hiemenz, P.C.; Rajagopalan, R.; P inciples o colloid and su ace chemis y, 3 d
Edi ion, Ma cel Dekke , Uni ed S a es o Ame ica, 1997.
Hi sch-Le ne , D.; Zhang, M.; Eliyahu, H.; Fe a i, M.E.; Wheele , C.J.;
Ba enholz, Y. (2005). E ec o "helpe lipid" on lipoplex elec os a ics,
Biochimica E Biophysica Ac a-Biomemb anes, 1714, 71.
Jones, R.L.; Lanie , A.C.; Keel, R.A.; Wilson, W.D. (1980). The e ec o ionic
s eng h on DNA-ligand unwinding angles o ac idine and quinoline
de i a i es., Nucleic Acids Resea ch, 8, 1613.
Kennedy, M.T.; Pozha ski, E.V.; Rakhmano a, V.A.; MacDonald, R.C. (2000).
Fac o s go e ning he assembly o ca ionic phospholipid-DNA
complexes ( ol 78, pg 1620, 2000), Biophysical Jou nal, 79, 1168.
Kim, J.Y.; Choung, S.; Lee, E.J.; Kim, Y.J.; Choi, Y.C. (2007). Immune ac i a ion
by siRNA/liposome complexes in mice is sequence- independen :
Lack o a ole o oll-like ecep o 3 signaling, Molecules and Cells,
24, 247.
Kyp , J.; Kejno ská, I.; Renčiuk, D.; Vo líčko á, M. (2009). Ci cula dich oism
and con o ma ional polymo phism o DNA, Nucleic Acids Resea ch,
37, 1713.
Lakowicz, J.R.; P inciples o Fluo escence Spec oscopy, 3 d Edi ion, Sp inge ,
Uni ed S a es o Ame ica, 2006.
Lu, X.J.; Olson, W.K. (2003). 3DNA: a so wa e package o he analysis,
ebuilding and isualiza ion o h ee‐dimensional nucleic acid
s uc u es, Nucleic Acids Resea ch, 31, 5108.

C
OMPACTION PROCESS OF
DNA
BY FLUORINATED LIPOSOMES
111
L , H.T.; Zhang, S.B.; Wang, B.; Cui, S.H.; Yan, J. (2006). Toxici y o ca ionic
lipids and ca ionic polyme s in gene deli e y, Jou nal o Con olled
Release, 114, 100.
Lyklema, J. (2009). Ques o ion-ion co ela ions in elec ic double laye s and
o e cha ging phenomena, Ad ances in Colloid and In e ace Science,
147-148, 205.
Ma ín-Molina, A.; Quesada-Pé ez, M.; Galis eo-González, F.; Hidalgo-Ál a ez,
R. (2003). P imi i e models and elec opho esis: An expe imen al
s udy, Colloids and Su aces A: Physicochemical and Enginee ing
Aspec s, 222, 155.
Ma ín-Molina, A.; Ma o o-Cen eno, J.A.; Hidalgo-Ál a ez, R.; Quesada-Pé ez,
M. (2008). Cha ge e e sal in eal colloids: Expe imen s, heo y and
simula ions, Colloids and Su aces A: Physicochemical and
Enginee ing Aspec s, 319, 103.
Ma ín-Molina, A.; Rod íguez-Beas, C.; Hidalgo-Ál a ez, R.; Quesada-Pé ez, M.
(2009). E ec o su ace cha ge on colloidal cha ge e e sal, Jou nal
o Physical Chemis y B, 113, 6834.
Ma ín-Molina, A.; Rod íguez-Beas, C.; Fa audo, J. (2010). Cha ge e e sal in
anionic liposomes: Expe imen al demons a ion and molecula o igin,
Physical Re iew Le e s, 104,
Ma ín-Molina, A.; Rod íguez-Beas, C.; Fa audo, J. (2012). E ec o Calcium
and Magnesium on Phospha idylse ine Memb anes: Expe imen s and
All-A omic Simula ions, Biophysical Jou nal, 102, 2095.
Ma y, R.; N'soukpoe-Kossi, C.N.; Cha bonneau, D.; Weine , C.M.; K eplak, L.;
Tajmi -Riahi, H.A. (2009). S uc u al analysis o DNA complexa ion
wi h ca ionic lipids, Nucleic Acids Resea ch, 37, 849.
112 C
OMPACTION PROCESS OF
DNA
BY FLUORINATED LIPOSOMES
Mengis u, D.H.; Bohinc, K.; May, S. (2009). Binding o DNA o zwi e ionic lipid
laye s media ed by di alen ca ions, Jou nal o Physical Chemis y B,
113, 12277.
Miyaha a, T.; Naka suji, H.; Sugiyama, H. (2012). Helical S uc u e and Ci cula
Dich oism Spec a o DNA: A Theo e ical S udy, The Jou nal o
Physical Chemis y A, 117, 42.
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.
Muñoz-Úbeda, M.; Rod íguez-Pulido, A.; Nogales, A.; Ma ín-Molina, A.;
Aica , E.; Junque a, E. (2010). E ec o lipid composi ion on he
s uc u e and heo e ical phase diag ams o DC-Chol/DOPE-DNA
lipoplexes, Biomac omolecules, 11, 3332.
Muñoz-Úbeda, M.; Rod íguez-Pulido, A.; Nogales, A.; Llo ca, O.; Quesada-
Pé ez, M.; Ma ín-Molina, A.; Aica , E.; Junque a, E. (2011). Gene
ec o s based on DOEPC/DOPE mixed ca ionic liposomes: A
physicochemical s udy, So Ma e , 7, 5991.
Nguyen, T.T.; Shklo skii, B.I. (2001a). Complexa ion o a polyelec oly e wi h
opposi ely cha ged sphe ical mac oions: Gian in e sion o cha ge,
Jou nal o Chemical Physics, 114, 5905.
Nguyen, T.T.; Shklo skii, B.I. (2001b). Complexa ion o DNA wi h posi i e
sphe es: Phase diag am o cha ge in e sion and een an
condensa ion, Jou nal o Chemical Physics, 115, 7298.
Pai a, D.; B ezesinski, G.; Pe ei a, M.d.C.; Rocha, S. (2013). Langmui
Monolaye s o Monoca ionic Lipid Mixed wi h Choles e ol o
Fluo ocholes e ol: DNA Adso p ion S udies, Langmui , 29, 1920.
Palme ini, C.A.; Came i, C.; Senna o, S.; Gaudino, D.; Ca lini, E.; Bo di, F.;
A ien i, G. (2006). Role o choles e ol, DOTAP, and DPPC in
C
OMPACTION PROCESS OF
DNA
BY FLUORINATED LIPOSOMES
113
p os asome/spe ma ozoa in e ac ion and usion, Jou nal o
Memb ane Biology, 211, 185.
Pawley, J.B.; Handbook o Biological Con ocal Mic oscopy, 3 d Edi ion,
Sp inge , Uni ed S a es o Ame ica, 2006.
Peula-Ga cía, J.M.; O ega-Vinuesa, J.L.; Bas os-González, D. (2010). In e sion
o Ho meis e Se ies by Changing he Su ace o Colloidal Pa icles
om Hyd ophobic o Hyd ophilic, The Jou nal o Physical Chemis y C,
114, 11133.
Piedade, J.A.P.; Mano, M.; de Lima, M.C.P.; O e skaya, T.S.; Oli ei a-B e ,
A.M. (2004). Elec ochemical sensing o he beha iou o
oligonucleo ide lipoplexes a cha ged in e aces, Biosenso s &
Bioelec onics, 20, 975.
Popa, I.; Gillies, G.; Papas a ou, G.; Bo ko ec, M. (2010). A ac i e and
Repulsi e Elec os a ic Fo ces be ween Posi i ely Cha ged La ex
Pa icles in he P esence o Anionic Linea Polyelec oly es, The
Jou nal o Physical Chemis y B, 114, 3170.
Pullmanno a, P.; Bas os, M.; Bai, G.Y.; Funa i, S.S.; Lacko, I.; De insky, F.;
Teixei a, J.; Uh iko a, D. (2012). The ionic s eng h e ec on he DNA
complexa ion by DOPC - gemini su ac an s liposomes, Biophysical
Chemis y, 160, 35.
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.
Ranjba , B.; Gill, P. (2009). Ci cula Dich oism Techniques: Biomolecula and
Nanos uc u al Analyses- A Re iew, Chemical Biology & D ug Design,
74, 101.
114 C
OMPACTION PROCESS OF
DNA
BY FLUORINATED LIPOSOMES
Ra ily, V.; San aella, C.; Vie ling, P.; Gulik, A. (1997). Phase beha io o
luo oca bon di-O-alkyl-glyce ophosphocholines and
glyce ophosphoe hanolamines and long- e m shel s abili y o
luo ina ed liposomes, Biochimica E Biophysica Ac a-Biomemb anes,
1324, 1.
Rodge , A.; No dén, B.; Ci cula Dich oism & Linea Dich oism, Ox o d
Uni e si y P ess, Uni ed S a es o Ame ica, 1997.
Rod iguez-Pulido, A.; Aica , E.; Llo ca, O.; Junque a, E. (2008). Compac ion
P ocess o Cal Thymus DNA by Mixed Ca ionic−Zwihe ionic
Liposomes: A Physicochemical S udy, The Jou nal o Physical
Chemis y B, 112, 2187.
Rod iguez-Pulido, A.; Ma in-Molina, A.; Rod iguez-Beas, C.; Llo ca, O.; Aica ,
E.; Junque a, E. (2009). A Theo e ical and Expe imen al App oach o
he Compac ion P ocess o DNA by Dioc adecyldime hylammonium
B omide/Zwi e ionic Mixed Liposomes, Jou nal o Physical Chemis y
B, 113, 15648.
San aella, C.; F éza d, F.; Vie ling, P.; Riess, J.G. (1993). Ex ended in i o blood
ci cula ion ime o luo ina ed liposomes, Febs Le e s, 336, 481.
Schindle , T.; No dmeie , E. (1997). The s abili y o polyelec oly e complexes
o Cal -Thymus DNA and syn he ic polyca ions: Theo e ical and
expe imen al in es iga ions, Mac omolecula Chemis y and Physics,
198, 1943.
Schneide , C.; Hanisch, M.; Wedel, B.; Jusu i, A.; Ballau , M. (2011).
Expe imen al s udy o elec os a ically s abilized colloidal pa icles:
Colloidal s abili y and cha ge e e sal, Jou nal o Colloid and In e ace
Science, 358, 62.
Senna o, S.; Bo di, F.; Came i, C.; Di Biasio, A.; Diociaiu i, M. (2005).
Polyelec oly e-liposome complexes: An equilib ium clus e phase
C
OMPACTION PROCESS OF
DNA
BY FLUORINATED LIPOSOMES
115
close o he isoelec ic condi ion, Colloids and Su aces a-
Physicochemical and Enginee ing Aspec s, 270, 138.
Shapi o, H.M.; P ac ical Flow cy ome y, 4 h Edi ion, John Wiley & Sons,
Uni ed S a es o Ame ica, 2003.
Sha ma, A.; Sha ma, U.S. (1997). Liposomes in d ug deli e y: P og ess and
limi a ions, In e na ional Jou nal o Pha maceu ics, 154, 123.
Simbe g, D.; Danino, D.; Talmon, Y.; Minsky, A.; Fe a i, M.E.; Wheele , C.J.;
Ba enholz, Y. (2001). Phase Beha io , DNA O de ing, and Size
Ins abili y o Ca ionic Lipoplexes, Jou nal o Biological Chemis y, 276,
47453.
Symie z, C.; Schneide , M.; B ezesinski, G.; Möhwald, H. (2004). DNA
alignmen a ca ionic lipid monolaye s a he ai /wa e in e ace,
Mac omolecules, 37, 3865.
Temple on, N.S.; Lasic, D.D.; F ede ik, P.M.; S ey, H.H.; Robe s, D.D.;
Pa lakis, G.N. (1997). Imp o ed DNA: liposome complexes o
inc eased sys emic deli e y and gene exp ession, Na u e
Bio echnology, 15, 647.
Tian, W.-d.; Ma, Y.-q. (2009). Molecula Dynamics Simula ions o a Cha ged
Dend ime in Mul i alen Sal Solu ion, The Jou nal o Physical
Chemis y B, 113, 13161.
Tian, W.-d.; Ma, Y.-q. (2010a). Complexa ion o a Linea Polyelec oly e wi h a
Cha ged Dend ime : Polyelec oly e S i ness E ec s,
Mac omolecules, 43, 1575.
Tian, W.-d.; Ma, Y.-q. (2010b). E ec s o alences o sal ions a a ious
concen a ions on cha ged dend ime s, So Ma e , 6, 1308.
Tian, W.-d.; Ma, Y.-q. (2011). Coa se-g ained molecula simula ion o
in e ac ing dend ime s, So Ma e , 7, 500.

116 C
OMPACTION PROCESS OF
DNA
BY FLUORINATED LIPOSOMES
Vo líčko á, M.; Kejno ská, I.; Bednářo á, K.; Renčiuk, D.; Kyp , J. (2012).
Ci cula Dich oism Spec oscopy o DNA: F om Duplexes o
Quad uplexes, Chi ali y, 24, 691.
Xu, Y.H.; Hui, S.W.; F ede ik, P.; Szoka, F.C. (1999). 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).
Uncon en ional U-shaped con o ma ion o a bolaamphiphile
embedded in a syn he ic hos , Chemical Communica ions, 46, 4091.
Be chel, M.; Mé iadec, C.; Lemièg e, L.; A zne , F.; Je ic, J.; Ben egnu, T.
(2009). Sup amolecula s uc u es based on new bolaamphiphile
molecules in es iga ed by small angle and wide angle X- ay sca e ing
and pola ized op ical mic oscopy, Jou nal o Physical Chemis y B,
113, 15433.
B unelle, M.; Polido i, A.; Denoyelle, S.; Fabiano, A.S.; Vuillaume, P.Y.;
Lau en -Lewandowski, S.; Pucci, B. (2009). A s uc u e-ac i i y
in es iga ion o hemi luo ina ed bi unc ional bolaamphiphiles
designed o gene deli e y, Comp es Rendus Chimie, 12, 188.
Ca uso, F.; Lich en eld, H.; Dona h, E.; Möhwald, H. (1999). In es iga ion o
Elec os a ic In e ac ions in Polyelec oly e Mul ilaye Films:  Binding
o Anionic Fluo escen P obes o Laye s Assembled on o Colloids,
Mac omolecules, 32, 2317.
Cosa, G.; Focsaneanu, K.S.; McLean, J.R.N.; McNamee, J.P.; Scaiano, J.C.
(2001). Pho ophysical P ope ies o Fluo escen DNA-dyes Bound o
Single- and Double-s anded DNA in Aqueous Bu e ed Solu ion,
Pho ochemis y and Pho obiology, 73, 585.
C ook, K.; S e enson, B.J.; Dubouche , M.; Po eous, D.J. (1998). Inclusion o
choles e ol in DOTAP ans ec ion complexes inc eases he deli e y
B
OLAAMPHIPHILE
-
BASED VECTORS
135
o DNA o cells in i o in he p esence o se um, Gene The apy, 5,
137.
Dakwa , G.R.; Hammad, I.A.; Popo , M.; Linde , C.; G inbe g, S.; Heldman, E.;
S epensky, D. (2012). Deli e y o p o eins o he b ain by
bolaamphiphilic nano-sized esicles, Jou nal o Con olled Release,
160, 315.
Deleu, M.; Damez, C.; Ga a d, S.; No , K.; Paquo , M.; Bouquillon, S. (2011).
Syn hesis and physico-chemical cha ac e iza ion o bolaamphiphiles
de i ed om alkenyl d-xylosides, New Jou nal o Chemis y, 35, 2258.
Dolle, C.; Mag one, P.; Ri a, S.; Amb osi, M.; F a ini, E.; Pe uzzi, N.; Nos o,
P.L. (2011). Symme ic and asymme ic bolaamphiphiles om
asco bic acid, Jou nal o Physical Chemis y B, 115, 11638.
Dona h, E.; Sukho uko , G.B.; Ca uso, F.; Da is, S.A.; Mohwald, H. (1998).
No el hollow polyme shells by colloid- empla ed assembly o
polyelec oly es, Angewand e Chemie-In e na ional Edi ion, 37, 2202.
Eliyahu, H.; Joseph, A.; Schillemans, J.P.; Azzam, T.; Domb, A.J.; Ba enholz, Y.
(2007). Cha ac e iza ion and in i o pe o mance o dex an-
spe mine polyplexes and DOTAP/choles e ol lipoplexes adminis e ed
locally and sys emically, Bioma e ials, 28, 2339.
Fle che , S.; Ahmad, A.; Pe ouzel, E.; He on, A.; Mille , A.D.; Jo gensen, M.R.
(2005). In Vi o S udies o Dialkynoyl Analogues o DOTAP
Demons a e Imp o ed Gene T ans e E iciency o Ca ionic
Liposomes in Mouse Lung, Jou nal o Medicinal Chemis y, 49, 349.
Fuh hop, J.-H.; Wang, T. (2004). Bolaamphiphiles, Chemical Re iews, 104,
2901.
Fuh hop, J.H.; F i sch, D. (1986). Bolaamphiphiles o m ul a hin, po ous and
unsymme ic monolaye lipid memb anes, Accoun s o Chemical
Resea ch, 19, 130.