Design, elabo a ion,
cha ac e iza ion and e alua ion
o niosome o mula ions o
gene deli e y o e ina and b ain
Mohamed Ahmed Ahmed Mashal
Vi o ia-Gas eiz 2020
NanoBioCel
G upo de Mic o y Nano Tecnologías,
Bioma e iales y Células
Design, elabo a ion,
cha ac e iza ion and e alua ion o
niosome o mula ions o gene
deli e y o e ina and b ain
Mohamed Ahmed Ahmed Mashal
NanoBiocel G oup, Labo a o y o Pha maceu ics
Uni e si y o he Basque Coun y (UPV/EHU)
Facul y o Pha macy
Vi o ia-Gas eiz, 2020
(cc)2020 MOHAMED AHMED AHMED MASHAL (cc by 4.0)
Design, elabo a ion,
cha ac e iza ion and e alua ion o
niosome o mula ions o gene
deli e y o e ina and b ain
Mohamed Ahmed Ahmed Mashal
NanoBiocel G oup, Labo a o y o Pha maceu ics
Uni e si y o he Basque Coun y (UPV/EHU)
Facul y o Pha macy
Vi o ia-Gas eiz, 2020

ACKNOWLEDGMENTS
Fi s and o he g ea es ex en , p aises and hanks o he Allah, he Almigh y, o
His showe s o blessings du ing he way o my esea ch wo k
.
I is said ha “I you' e no hank ul o people, you' e no hank ul o God”. The e o e,
I ha e o say ha he comple ion o his hesis would ha e been impossible wi hou he
lo e, suppo , guidance and encou agemen o many indi iduals.
I would like o begin by exp essing my deepes g a i ude o my dea es supe iso P o .
D . Jose Luis Ped az o gi ing me he oppo uni y o wo k on his hesis in his
labo a o y. He augh me he p eciseness in science and he good p ac ice in he lab. I
eel mysel p i ileged o being able o wo k unde his expe ienced supe ision owa ds
accomplishing his esea ch success ully. Hones ly, I’ e always conside ed p o . D .
Ped az and Angela as my amily in Spain. D . Ped az, I can' hank you enough o all
ha you ha e done o me!
I is wi h g ea pleasu e ha I am exp essing my hanks and app ecia ions o my dea es
supe iso , P o . D . Gus a o Pu as, o whom I am immensely indeb ed. I uly hank
him o being so en husias ic abou he wo k, o his g ea scien i ic guidance,
e iewing he hesis manusc ip and o his decen cons uc i e commen s.
I can’ exp ess enough hanks o my soul ma e, wi e and co-wo ke , D . Noha A ia, o
wha no wo ds can desc ibe. Noha, wi hou you dedica ions, suppo , and pa ience,
ob aining my Ph.D. would ha e been o ally impossible.
Resea ch is an exci ing jou ney o disco e y. Thus, I ex end my g a i ude o all co-
au ho s o hei since e in aluable suppo . The human, alongside wi h he scien i ic
suppo om all membe s o he eam was a con inuous eeling h oughou he whole
jou ney in he lab.
Las ly, I own my wa mes hanks o my dea ones, my Mom (Layla) and all my amily
membe s.
ACKNOWLEDGMENT FOR THE FINANCIAL SUPPORT
This p ojec was suppo ed by he Basque Coun y Go e nmen (CGIC10/172),
Spanish Minis y o Educa ion (G an CTQ2017-84415-R, MAT2015-69967-C3-1R),
he Gene ali a de Ca alunya (2014/SGR/624), and he Ins i u o de Salud Ca los III
(CB06_01_0019, CB06_01_1028). The au ho s also wish o hank he in ellec ual and
echnical assis ance om he ICTS “NANBIOSIS”, mo e speci ically by he D ug
Fo mula ion Uni (U10) o he CIBER in Bioenginee ing, Bioma e ials, and
Nanomedicine (CIBER-BBN) a he Uni e si y o Basque Coun y (UPV/EHU).
Technical and human suppo p o ided by SGIke (UPV/EHU) is g a e ully
acknowledged.
ACKNOWLEDGMENT TO THE EDITORIALS
Au ho s would like o hank he edi o ials o g an ing pe mission o euse hei
p e iously published a icles in his hesis.
Mashal Mohamed, e al. "Re inal gene deli e y enhancemen by lycopene inco po a ion
in o ca ionic niosomes based on DOTMA and polyso ba e 60."Jou nal o Con olled
Release 254 (2017): 55-64.
Mashal Mohamed, e al. "Non- i al ec o s based on ca ionic niosomes as e icien
gene deli e y ehicles o cen al ne ous sys em cells in o he b ain."In e na ional
jou nal o pha maceu ics 552.1-2 (2018): 48-55.
Mashal, Mohamed, e al. "Gene deli e y o he a e ina by non- i al ec o s based on
chlo oquine-con aining ca ionic niosomes."Jou nal o Con olled Release 304 (2019):
181-190.
Man canno emake himsel wi hou su e ing,
o he is bo h he ma ble and he sculp o .
"Man, The Unknown" Alexis Ca el
Chap e 1
4
(4). This was a majo achie emen and allowed long s e ches o DNA o be apidly
and accu a ely sequenced. In 1983, he polyme ase chain eac ion (PCR) echnique
was disco e ed by Ka y Mullis, a b eak h ough ha enabled scien is s o apidly
ampli y DNA (5).
1.1.2. Clinical ials and comme cial p oduc s o gene he apy
A e decades o esea ch, cell and gene he apies a e mo ing om bench lab o
bedside. Since he yea 1989 ( i s gene he apy clinical ial), mo e han 2600 clinical
ials o gene he apy ha e been app o ed globally ill he yea 2017 (Figu e 1) (6).
Figu e 1. The numbe o gene he apy clinical ials app o ed wo ldwide 1989-2017
(6).
In 1990 he 4-yea -old DeSil a wi h se e e combined immune de iciency
(SCID) became he i s pa ien o unde go gene he apy in he Uni ed S a es. This ial
was au ho ized by he NIH Recombinan DNA Ad iso y Commi ee (RAC) and he
Food and D ug Adminis a ion (FDA). Func ional adenosine deaminase (ADA) genes
144
12814
37 38
67 51
82 68
117
9610898 85101112
117
90
120
81 92 87102
125
135
169
118
132
0
20
40
60
80
100
120
140
160
180

Chap e 1
5
we e ans e ed by e o i al ec o s in o he cul u ed T cells. The T lymphocy es
wi h he co ec ed gene we e ein used back in o he pa ien abou 12 days a e blood
was d awn. Also, a he NIH Clinical Cen e in1991, Cyn hia a 9-yea s-old gi l
unde wen ano he ial. Un o una ely, in 1999 he 18-yea -old Jesse Gelsinge wi h
inhe i ed enzyme de iciency was he i s ic im o gene he apy a e 4 days o
injec ion wi h a gene ically al e ed adeno i us in o his li e . This was a di ec eason
o se e e handicap in he esea ch ield o his echnology (7). The ield o gene
he apy was shaken again in 2002, when a h ee-yea -old boy wi h SCID ea ed in a
F ench ial de eloped leukemia. The e o e, gene he apy clinical ials we e hal ed
again (8). Ne e heless, Gendicine ( ecombinan human p53 adeno i us) was
app o ed in 2003 by he China Food and D ug Adminis a ion (CFDA) as a i s gene
he apy p oduc o ea head and neck cance (9). In 2007, B i ish doc o s pe o med
he wo ld’s i s gene he apy ope a ion o ea Lebe Congeni al Amau osis due o
RPE65 mu a ions ia ocula sub e inal injec ion o adeno-associa ed i us (AAV) gene
ec o (10). In 2012, he i s gene he apy d ug app o ed by he Eu opean Medicines
Agency (EMA) was Glybe a (alipogene ipa o ec). Glybe a is an AAV ec o
enginee ed o exp ess lipop o ein lipase o he ea men o lipop o ein lipase
de iciency (11).
Figu e 2. Dis ibu ion coun ies o app o al gene he apy d ugs (12)
Chap e 1
6
The yea o 2017 is conside ed he dis inguished s a ing poin o gene he apy
in he Uni ed S a es. In Augus 2017, kym iah® was app o ed by he FDA as he i s
gene he apy p oduc o be ma ke ed o ea men o acu e lymphoblas ic leukemia. In
he same yea , Yesca a®, was app o ed by FDA o ea ing la ge B-cell lymphoma.
Fu he mo e, by Decembe 2017, he FDA-app o ed Lux u na® became he i s in i o
gene he apy d ug o ea Lebe congeni al amau osis LCA (a a e inhe i ed eye
disease). Recen ly, Zolgensma® became he i s gene he apy app o ed by FDA in
2019 o ea child en less han wo yea s o age wi h spinal muscula a ophy (SMA),
a leading he edi a y cause o in an mo ali y.
Figu e 3. Timeline o gene he apy de elopmen (12)
Such ecen ly app o ed d ugs o human use based on gene, along wi h ad ances on
e olu iona y genome edi ing echnologies, sugges ha gene he apy can become a
egula medical op ion in o he clinical p ac ice.
Chap e 1
7
One o he o gans ha is conside ed an en icing gene he apy a ge is he eye,
because o i s accessibili y and i s immune p i ilege. The ision p ocess is highly
complex, and equi es coo dina ion o nume ous componen s in bo h eye and b ain.
Mo eo e , he e ina o he eye ep esen s a pa o he cen al ne ous sys em (CNS)
and is ac ually a b ain issue. Al hough he RPE65 gene he apy ( ia i al ca ie s) has
pa ed he way o he ea men o e inal diseases. Some o he genes a e oo la ge o
be ca ied in o he e ina ia i al ca ie s, such as he gene causing S a ga d ’s
he edi a y maculopa hy.
The Re inos a ® is a gene he apy ha is cu en ly in phase I clinical ial o he
we sub ype o age- ela ed macula degene a ion (AMD). I inhibi s blood essel
g ow h by exp essing angios a in and endos a in p o eins. On he o he side, he sFLT
gene (ca ied on AAV ec o ) was injec ed sub e inal o block he ascula endo helial
g ow h ac o (VEGF) (13). Table 1 depic s some gene he apy clinical ials o eye
diso de s a di e en s ages. In a CNS clinical ial (NCT01454596), he gene o
epide mal g ow h ac o ecep o (EGFR III) was inco po a ed in e o i us o ea
pa ien s wi h glioblas oma. A e o i al ec o anspo ing a chime ic an igen ecep o
(CAR) o he EGFR III umo an igen, can be used o media e gene ic ans e o CAR
wi h high e iciency (14). Table 2 elabo a es mo e on he gene he apy clinical ials o
CNS diso de s.
Table 1. Gene he apy clinical ials on eye diseases (15).
Chap e 1
8
Table 2. Gene he apy clinical ials on CNS diseases (15).
1.1.3. Gene he apy s a egies
Cu en ly, a ious gene he apy s a egies do exis . Such s a egies could be
ca ego ized in o: (1) gene eplacemen o he monogenic diseases, (2) gene addi ion o
ea acqui ed diseases, (3) gene edi ing o in oduce a ge ed changes in hos genome,
and (4) al e a ion o gene exp ession by a ge ing RNA. The ea men may ake place
ou side o he body (ex i o) o inside he body (in i o). Modi ied i uses o o he
ec o s a e used as gene deli e y sys ems To deli e he gene in o he genome in o he
cells.
The signi ican po en ial o using plasmids o gene he apy has been ecognized
since 1990. Selec ing he igh composi ion o a plasmid is undamen al o ensu e he
success o gene he apy. The e a e many ac o s and elemen s o be conside ed when
choosing he plasmid backbone such as: cloning o exp ession sa e y, plasmid size,
an ibio ic esis ance, es ic ion si es in mul iple cloning si e (MCS), p omo e ,
e mina o , ibosome binding si e (RBS) sequence, o p o ein modi ica ions (adding a
ag o a usion p o ein o he plasmid o u he unde s and he unc ion o a speci ic
gene) (16). Bo h igu e 4 and able 3 desc ibe di e en elemen s o he plasmid.
Chap e 1
9
Figu e 4. A ep esen a i e map o he main ea u es o a plasmid ec o . (17)
Table 3. Plasmid elemen s and hei desc ip ion. (17)
Plasmids a e simple and cheape o make, ship and s o e compa ed wi h i al
and RNA-based ec o s and ha e a much longe shel li e. The modula na u e o
plasmids also allows enables simple molecula cloning, making hem easy o
manipula e and design o he apeu ic use. In addi ion, he plasmids can be dis ibu ed
epea edly, unlike i uses. Mos plasmid DNA p epa a ions include many opological
plasmid a ia ions, including supe coiled ( he p e e ed opology), bu also he
undesi able linea and open ci cula o ms o he plasmid. In o de o deli e hei

Chap e 1
10
payload, Plasmids equi e ec o s, physical o ces, o ad anced modi ica ions o
up ake and nuclea localiza ion. Since plasmids a e non- eplica ing episomes, he
exp ession o ansgenes is ansien and dilu ed by cell di ision. The dinucleo ides o
unme hyla ed cy osine-phospha e-guanine (CpG) a e mo e widesp ead in bac e ial
DNA han in mammalian DNA. They ha e he abili y o be ecognized by he
mammalian immune sys em ia oll-like ecep o (TLR)-9, and a e po en ially
p ecipi a ing no only in ansgene silencing, bu also in immune esponse (18).
RNA p ecu so s a e essen ial a ge s in gene ic he apies. An isense
oligonucleo ides (ASOs) a e single-s anded (ss) DNA o RNA sequences ha can be
used o silencing o e exp essed p o eins in oxic “gain o unc ion” diseases. They can
a ge RNA o deg ada ion, p e en ing he ansla ion o a speci ic RNA in o p o ein
and al e ing he splicing o p e-mRNA. ssDNA complemen a y o mRNA could be used
o block he ansla ion o speci ic mRNA (19).
Ap ame s a e sho , single-s anded DNA o RNA (ssDNA o ssRNA)
molecules ha can bind o a speci ic a ge , including p o eins, pep ides, ca bohyd a es,
small molecules, oxins, and e en li ing cells. Ap ame s a e selec ed om a la ge
oligonucleo ide lib a y h ough a p ocess called SELEX (Sequen ial E olu ion o
Ligands by Exponen ial En ichmen ) (20).
Recen ly, he gene edi ing ool CRISPR/Cas9 (Clus e ed Regula ly In e spaced
Sho Palind omic Repea s/ CRISPR-associa ed p o ein 9) was used ( igu e 5). Cas9 is
an RNA-guided DNA endonuclease enzyme ha uses CRISPR sequences as a guide o
iden i y and clea e speci ic s ands o DNA complemen ing he CRISPR sequence in
o de o media e genome al e a ion wi h high p ecision. Unlike he p e ious gene
edi ing echniques such as T ansc ip ion Ac i a o s-Like E ec i e Nucleases
(TALENS) his echnology is simple, easy o use and inexpensi e. Howe e , hey ha e
limi a ions, such as na ow a ge ing ange and po en ial o o - a ge mu agenesis
(21).
Bu wi h highe p ecision, one o he app oaches based on he CRISPR
echnique is p ime edi ing echnology ool, which bo h speci ies he a ge loca ion and
encodes he desi ed edi . The p ime echnique w i es new gene ic in o ma ion di ec ly
Chap e 1
11
in o a speci ied DNA si e using a ca aly ically impai ed Cas9 endonuclease used o an
enginee ed e e se ansc ip ase, p og ammed wi h a p ime edi ing guide RNA
(pegRNA). I ew i es DNA by cu ing jus one s and o add, dele e, o subs i u e base
pai s ha can edi mo e gene ic mu a ion o ms han genome-edi ing app oaches like
CRISPR-Cas9 (22).
RNA in e e ence (RNAi) is o mula ed and p esen ed as a small double-s anded (ds)
RNA in ended o down- egula e i s a ge ansc ip (i.e., mRNA) and p o ein.
None heless, o long- e m knockdown, a mo e obus s a egy is o use ano he RNAi
ou e, by deli e ing syn he ic mic oRNA (miRNA) (24). Addi ionally, sho in e e ing
RNAs (siRNAs) can down egula e a selec ed mRNA gene ha appea s o deg ade
h ough he RNA-in e e ing silencing complex (RISC). The pla o ms siRNA and
ASO a e bo h nucleic acids con aining an isense s and designed o iden i y a a ge
mRNA. Though, ASOs ha e one s and while siRNAs ha e wo, which can educe cos
and simpli y deli e y (25).
In 2018, Alnylam launched he ONPATTRO® (pa isi an) d ug which has
been app o ed by he FDA as he i s siRNA-based he apy o he a e he edi a y
disease ans hy e in-media ed amyloidosis in adul pa ien s (26).
Chap e 1
12
Figu e 5. CRISPR/Cas9-media ed DNA clea age and simpli ied epai mechanism
(23).
1.2.Gene deli e y ec o s
Cu en gene deli e y ehicles, namely ec o s, a e classi ied mainly in o wo
classes: he i al and he non- i al ec o s.
1.2.1.Vi al ec o s
Vi uses a e a ac i e gene-deli e y ehicles due o hei abili y o e icien
nucleic acid deli e y o speci ic cell ype while escaping hos cell immune su eillance.
Mo eo e , hey may exploi he cellula machine y o encou age hei eplica ion (27).
Vi al deli e y sys em is based on a i us wi h nucleic acid coa ed by capsid p o ein and
in many cases u he by an en elope s uc u e. One o mo e i al s uc u al genes a e
dele ed o deac i a e he i us om sp ead in he hos o ganism (28). Vi al ec o s had
been designed o empo a y sho - e m and pe manen long- e m exp ession, and can
be con eyed by bo h RNA and DNA i uses wi h ei he single-s anded o double-
Chap e 1
13
s anded genomes (29). Howe e , many disad an ages and sa e y issues a e ela ed o
he use o i al ec o s such as complex and expensi e p oduc ion, limi ed ca ying
capaci y, b oad opism, oncogenici y, mu agenici y, immunogenici y and
in lamma o y esponses (30). Recen ly, chime ic i al ec o s ha e been de eloped in
a ial o o e come he limi a ions o each i al species, by combining sui able ea u es
o wo o mo e di e en i uses in o one (31).
The e o i al capsid is an en eloped shell o p o ein wi h size o 80-100 nm. I
has an a e age packaging capaci y o 8 kb. Re o i al ec o in eg a es in he hos cell
genome, by using e e se ansc ip ase in a s able and pe manen manne . Replica ion-
compe en and eplica ion-de ec i e a e he wo ypes o e o i al ec o s. Re o i us
a e mainly applied in s udies o issue epai and enginee ing due o hei capabili y o
in ec ing di iding cells wi hou de eloping any immunogenic i al p o eins (32).
Len i i uses, sub ype o e o i us, a e able o in eg a e in o non-di iding cells.
The len i i al genome is simila o o he e o i uses; howe e , i con ains six o he
genes - wo egula o y genes and ou accesso y genes- ha code o p o eins essen ial
o i al eplica ion, binding, in ec ion, and elease. Len i i us common example is he
human immuno- de iciency i us ype 1 (HIV-1). Len i i al ec o s seem o be less
mu agenic han hei e o i al coun e pa s (33).
Adeno i uses a e icosahed al, non-en eloped, ds DNA i uses wi h a size o 70
nm, and a genome size o abou 36 kb. They a e he mos widely used i al ec o s able
o o e come he limi a ions o o he i al ec o s as he e o i us Among he 50
di e en se o ypes o adeno i us, he ypes 2 and 5 a e he mos equen ly used ones
(34). The packaging capaci y o dsDNA adeno i uses is abou 7.5 kb o o eign DNA
wi h sho - e m episomal exp ession wi h b oad ange o hos cells. In addi ion, he e
is a possibili y o hyb idize adeno i us ec o s wi h sleeping beau y ansposase sys em
o ch omosomal in eg a ion o wi h CRISPR/Cas9 nuclease o gene edi ing (35).
The AAV is a small (22 nm in diame e ) nonpa hogenic pa o i us wi h a non-
en eloped, icosahed al capsid. I s genome is composed o a linea , ss DNA. I is used
Chap e 1
20
Solid lipid nanopa icles (SLN) we e p esen ed in 1990 as a subs i u e deli e y
sys em o liposomes, emulsions and polyme ic nanopa icles. SLNs a e composed o
solid a which is dispe sed in an aqueous phase in he p esence o su ac an s o
enhance i s s abili y. The p ope selec ion o lipids and su ac an s can a ec he pa icle
size, s abili y, loaded molecules and beha io s o elease. Thei lipid componen s a e
solid a bo h body and ambien empe a u e (57).
Nanos uc u ed lipid ca ie s (NLCs) a e sphe ical s uc u es wi h a mixed
solid and liquid ma ix, ha ing an aqueous co e su ounded by a lipid bilaye . The e
a e h ee majo ypes o NLC: ca ionic, neu al, and a ge ing-modi ied NLC. Ca ionic
NLCs can be used as ca ie s o nega i ely cha ged subs ance, including p o eins,
polypep ides, oligonucleo ides, RNAs and DNAs (58) NLC ha e many clinical
applica ions because o supe io biocompa ibili y, high biodeg adabili y and low
immunogenici y. They a e used in he deli e y o nucleic acids including dis inc
miRNA molecules o cance gene he apy.
Niosomes a e sel -assembled non-ionic su ac an esicles. They a e composed
o h ee main componen s: (1) a non-ionic su ac an such as polyso ba es; (2) a neu al
helpe lipid such as choles e ol, squalene and lycopene; (3) a ca ionic lipid such as
DOTMA. The inco po a ion o non-ionic molecules in niosomes educes he
undesi able oxici y o ca ionic lipids showing be e cellula iabili y p o iles
compa ed o hei co esponding anionic o ca ionic coun e pa s (59). Niosomes ha e
i s eme ged as a esicle deli e y sys em in he 70s in he ield o cosme ics indus y.
Thanks o hei capabili y o encapsula e bo h hyd ophobic and hyd ophilic d ugs,
niosomes a e epo ed as po en ial ca ie s o he deli e y o d ugs such as doxo ubicin,
accines, insulin, siRNA. They ha e many applicable he apeu ic e ec s (e.g. an i-
Alzheime , an i-cance , an i-oxidan , an i-diabe ics and an i-mic obial) and can be
adminis a ed ia a ious me hods, as in a enously, o ally (e.g., Flu bip o en); ocula
(e.g., Chlo amphenicol, Ace azolamide, Fluconazole), and opically (e.g.,
E y h omycin, Minoxidil, Ro ecoxib) (60). Niosomes a e highly s able, ye sligh ly
leakie han liposomes. In compa ison o liposomes, niosomes can be o mula ed a a
lowe cos , longe s abili y and less oxici y. Mo eo e , esea che s highligh ed on he

Chap e 1
21
biocompa ible, biodeg adable pha maceu ics and low immunogenic ea u es o hei
componen s (non-ionic su ac an s, helpe lipids as well as cha ged molecules).
Niosomes ha e been widely used as oligonucleo ide ca ie s. The o mula ion o
niosomes, by he me hod o sol en emulsi ica ion-e apo a ion echnique, managed o
deli e pCMS-EGFP plasmid o he e ina and b ain. The esul s p o ed ha niosomes
could p o ec DNA om deg ada ion and in oduced good a icking pa e n wi h good
chemical and physical s abili y and ela i ely smalle sizes. In addi ion, niosomes can
also be used as ec o s in DNA accines which p o ide a simple, s able and cos -
e ec i e solu ion compa ed wi h liposomes. Niosomes can also assis as a deli e y
sys em o a ge ing s em cells (60).
Lipopolysomes a e mul i unc ional nanoca ie s composed by combining
polyme somes and liposomes. Lipopolysomes ha e he abili y o simul aneous
encapsula ion o hyd ophobic and hyd ophilic molecules (61). The ec o esul ed by
co‐ o mula ion o plasmid DNA and lipopolysomes is called lipopolyplexes. In non-
i al gene deli e y sys ems his e m (lipopolyplexes) conclude a di e se componen o
lipids and polyme s co- o mula ed wi h gene ic ma e ials. One ad an age o
lipopolyplexes ehicles is ha hey ha e he po en ial o be a ge ed o speci ic cell
ypes by a aching pep ide a ge ing ligands on he su ace, hus inc easing bo h he
ans ec ion e iciency and selec i i y o disease a ge s such as cance cells (62).
Chap e 1
22
Figu e 9. Di e en ec o s (A) and ec o /DNA complexes (B) con aining ca ionic
lipid.
1.4.Gene deli e y ba ie s
On hei way o he a ge cells in i o, gene deli e y sys ems mus ci cum en
se e al ex acellula and in acellula ba ie s (po ayed in igu e 10). The e o e, he
design o inno a i e op imal non- i al ec o s is a g ea challenge o su pass hese
hu dles.
1.4.1. Ex acellula ba ie s
Wha e e he adminis a ion ou e is (e.g., inhala ion, in amuscula injec ion,
ga age, in a ascula injec ion, o al, e c.), gene deli e y ec o will be ine i ably in
con ac wi h he ex acellula en i onmen . Basically, hese ex acellula ba ie s
Chap e 1
23
include lipid bilaye memb ane, many blood componen s and endo helial ba ie s.
Addi ional ex acellula ba ie s need o be o e come by he ehicle when speci ic
issues need o be a ge ed, such as he b ain, he eye o he lungs (37). The con ol o
unwan ed immune esponses is he way o success o gene he apy s a egies. Gene
ec o s a e po en ially able o igge immune esponses as lymphocy es. Mo eo e ,
enzymes such as lipases and nucleases can deg ade he nano- o mula ion and he
gene ic payload and he e o e in e e e wi h ans ec ion e iciency (63). The i al and
non- i al ec o s ha e been shown o induce an immune esponse. Howe e , his
ac i a ion has been mos associa ed wi h i al ec o s. The injec ion o ca ionic
lipoplexes in he ci cula ion cause he elease o TNFα and IFNγ in o he se um as
in lamma o y esponse chemokines. This may be as a esul o unme hyla ed CpG
mo i s on he plasmid DNA and he subsequen ecogni ion by TLRs (64).Ex acellula
ba ie o be o e come s ongly depend on he o gan o be ea ed as well as he ou e
o adminis a ion.
In he sys emic ci cula ion, blood lows o e e y o gan and issue in he body.
Gene ally, in a enous adminis a ion is he mos s udied ou e o adminis a ion o
non- i al gene deli e y sys ems. Once gene ehicles a e in oduced in o he ci cula ion,
hey a e subjec o enzyma ic deg ada ion, se um p o eins inac i a ion, complemen -
media ed clea ance and e iculoendo helial sys em ecogni ion (66). Fo med blood
elemen s (e y h ocy es, leukocy es, and pla ele s) and se um p o eins ( albumin,
immunoglobulins, and ib onec in) ha e a nega i e su ace cha ge which in e ac s wi h
he ne posi i e cha ge o non- i al ec o /DNA complex leading o agg ega ion o
dissocia ion o he complex (67).
The deli e y o non- i al gene ec o o he lung is usually hampe ed by he
pulmona y a chi ec u e, he p esence o espi a o y sec e ions (mucus and lung
su ac an ), he clea ance mechanisms, and he ac i a ion o he immune sys em.
Acco ding o he lung disease (e.g., cys ic ib osis, as hma, emphysema and lung
cance ), he a ge cells can a y om epi helial cells, al eola cells, mac ophages,
espi a o y s em cells o endo helial cells (68). The espi a o y sec e ions bind o he
Chap e 1
24
complexes and s e ically obs uc hei way o he a ge cells limi ing hei di usions
and e ec i eness.
Figu e 10. Ex acellula and in acellula ba ie s, adap ed om (65)
The blood-b ain ba ie (BBB) ep esen s a huge obs acle upon sys emic
deli e y o non- i al ec o /DNA complexes in o he b ain. BBB is composed o b ain
mic o ascula endo helial cells, pe icy es, as ocy es, igh junc ions, and basal laminae
(69). Many app oaches exploi he ecep o -media ed up ake o molecules such as
ans e in, lac o e in and insulin o c oss he BBB, since ecep o s o hose molecules
Mac ophage
.
Endonucleases
Sys emicdeli e y
Ex acellula ba ie s
Plasmap o einsComplex
Nucleus
Nuclea po e
T ansc ip ion
Endosome
Endocy osis
DNAplasmid
In acellula ba ie s
Ex acellula ma ix
Ex a asa ion
Chap e 1
25
a e exp essed on many cell ypes, including neu ons and he capilla y endo helial cells
o he BBB. Ano he app oach uses pep idomime ic monoclonal an ibodies (known as
a molecula T ojan ho se) o a ge speci ic ecep o s on he BBB and induce ecep o -
media ed anscy osis o he non- i al ec o in o he CNS. O he s a egy includes
ansien mechanical dis up ion o he BBB and RNAi-media ed knockdown o igh
junc ion p o eins (70). Local adminis a ion o he b ain, ei he by injec ion o by
in usion is in es iga ed in many p e-clinical s udies. In anasal deli e y is ano he non-
in asi e means o deli e non- i al gene ca ie s o he b ain, wi h he abili y o pass
he BBB and ans ec and exp ess he encoded p o eins (71).
Wi h ega d o he eye, i is a highly compa men alized and immune-p i ileged
o gan ha o e s in e es ing ad an ages as a gene he apy a ge (72). Howe e , ele an
biological ba ie s such as co nea, scle a, aqueous humo , blood- e inal ba ie s (BRB),
cho oidal and conjunc i al blood low, lympha ic clea ance, and ea dilu ion need o
be deeply conside ed. The BRB, which is composed o igh epi helial junc ions, limi s
he deli e y o non- i al ec o /DNA complexes o he e ina ia sys emic
adminis a ion. Two s a egies we e sugges ed o o e come such BRB, (1) by using
ec o s smalle han 100 nm, and (2) by he use o ligand- a ge ed ec o s ha ecognize
speci ic ecep o s in he BRB (37).
1.4.2. In acellula ba ie s
Gene deli e y sys ems ha e o o e come many in acellula ba ie s ex ending
om cell su ace o nuclea en y o success ul gene he apy. The non- i al
ec o /DNA complex en e he cells ei he by ligand- ecep o binding in e ac ion
( ecep o -media ed endocy osis) o by cha ge-media ed in e ac ions wi h p o eoglycans
on cell memb anes. Thus, he esicles a e suscep ible o a cascade p ocesses o complex
up ake, endolysosomal escape, a icking o he nucleus, ehicle unpacking and
nuclea en y (73).
Being up aken in o cells is no he only limi ing ba ie , howe e i is one o
he mos limi ing s eps a ec ing non- i al ehicle e iciency. Endocy osis is a esicle-
media ed p ocess ha can be media ed by i e main endocy ic pa hways: cla h in-

Chap e 1
26
media ed endocy osis (CME), ca eolae-media ed endocy osis (C ME), cla h in-
ca eolae-independen endocy osis, mac opinocy osis and phagocy osis (74).
Lysosomes a e memb ane-bound in acellula o ganelles wi h an acidic pH (4-
5). They ha e an essen ial ole o deg ada ion and ecycling o mac omolecules
deli e ed by endocy osis, phagocy osis, and au ophagy (75). The abili y o many non-
i al ehicles o deli e nucleic acid e icien ly may be a ibu ed o hei s ong
bu e ing capaci y (pH anges om 5 o 7). Such s ong bu e ing capaci y p e en s he
acidi ica ion o endosomes by ac ing as ‘p o on sponges’ (48).
P oduc i e gene ans e equi es DNA o e en ually c oss he nuclea en elope
h ough nuclea po e complexes (NPCs) be o e ini ia ion o ansc ip ion. In he
absence o cell di ision, he in ac nuclea en elope impedes he en y o ca ie -
plasmid complexes (76). Va ious app oaches we e used o imp o e plasmids’ nuclea
a ge ing, such as: complexa ion o plasmids wi h pep ides, p o eins, ligands, polyme s,
and inclusion o ansc ip ion ac o -binding si es (77). As well, nuclea localiza ion
sequence (NLS) pep ides can be di ec ly bound o he DNA in o de o p omo e i s
anspo o he nucleus by he impo ins (78).
Chap e 1
27
Figu e 11. Mechanisms o cellula up ake o non- i al ec o /DNA complex.
1.5.S abili y o o mula ions
Chemical non- i al ec o s a e liquid o mula ions which upon s o age a e
suscep ible o s abili y p oblems. In gene al, nanopa icles a e ex emely uns able due
o hei high su ace ene gy and hey end o change hemsel es o eac wi h subs ances
o each a ela i ely s able s a e. Changes du ing s o age and/o shipping equi es
p epa a ion o eshly lipid/DNA complexes be o e e e y single use. Ne e heless, an
accep able s abili y is essen ial o pha maceu ical de elopmen and
comme cializa ion. Gene ally, physical s abili y a ec s he shel -li e o non- i al
ec o ha is ela ed o many pa ame e s, such as: uni o mi y o size dis ibu ion, PDI,
ze a po en ial, lamella changes, agg ega ions and/o usion (79). The change in such
pa ame e s may be conside ed as a unc ion o pH, empe a u e, bu e concen a ion,
ionic s eng h, s o age ime, e c. (80).
Mic opinocy osis Phagocy osis Cla h in- andca eolae-
independen 
endocy osis
Cla h in-dependen 
endocy osis
Ca eolae-dependen 
endocy osis
Cellmemb ane
Mac opinosome
Lysosome
Cla h incoa ed esicle
Phagosome
Ca eosome
La eendosome
Nucleus
Unpacking
Nuclea impo
Non- i al ec o /DNA
complex
Dynamin
Cla h in
Ca eolin
endosome
Chap e 1
28
The physical s abili y s udy upon s o age a di e en empe a u e (mainly a
25°C, 4°C, -20°C and -80°C) is one o he basic s abili y s udies. F eezing could al e
he memb ane s uc u e, and consequen ly he shape, o Lipo ec amine 2000® esicles
and enhance hei gene deli e y ac ion wi hou comp omising cell iabili y in many
cell lines (81). Gene ally, he changes in physical cha ac e is ics by eezing o esicles
can lead o dec ease o inc ease in ans ec ion e iciency. The s o age a subze o
empe a u e may be a good subs i u e o s o age a 4°C due o hyd oly ic deg ada ion
caused by excess bulk wa e o aqueous o mula ion leading o less s able o mula ion
on long- e m s o age. None heless, c yop ese a ion a -80°C was used o he s o age
o liposomes, i s use o DNA complexes may be unlogic as he addi ion o
c yop o ec an s as DMSO and suga s may a ec he ans ec ion e icacy and iabili y
o he DNA complexes. In addi ion, c yop ese a ion had a damaging e ec a he
molecula le els specially on he DNA molecules (82).
The biological s abili y o chemical non- i al ec o s e e s o he in e ac ion o
DNA wi h di e en componen s in he biological sys em. Be e unde s anding o hei
in e ac ions is essen ial o es ablish speci ic design c i e ia. The agg ega ion o DNA
by se um p o eins and deg ada ion by DNase enzymes a e essen ial obs acles in he
s abili y o DNA ec o s. In e es ingly, PEG coa ing ends o p o ide he p o ec ion o
DNA agains he se um deg ada ion haza ds (83).
1.6.Re e ences
1. Wi h T, Pa ke N, Ylä-He uala S. His o y o gene he apy. Gene.
2013;525(2):162-9.
2. F iedmann T, Roblin R. Gene he apy o human gene ic disease? Science.
1972;175(4025):949-55.
3. Kelly J TJ, Smi h HO. A es ic ion enzyme om Hemophilus in luenzae: II.
Base sequence o he ecogni ion si e. Jou nal o molecula biology. 1970;51(2):393-
409.
4. To omoch-Se a A, Ma quez MF, Ce an es-Ba agán DE. Sange sequencing
as a i s -line app oach o molecula diagnosis o Ande sen-Tawil synd ome.
F1000Resea ch. 2017;6.
5. Ba le JM, S i ling D. A sho his o y o he polyme ase chain eac ion. PCR
p o ocols: Sp inge ; 2003. p. 3-6.
6. Ginn SL, Amaya AK, Alexande IE, Edels ein M, Abedi MR. Gene he apy
clinical ials wo ldwide o 2017: An upda e. The jou nal o gene medicine.
2018;20(5):e3015.
Chap e 1
29
7. Ma shall E. Gene he apy dea h p omp s e iew o adeno i us ec o . Science.
1999;286(5448):2244-5.
8. Check E. Second cance case hal s gene- he apy ials. Na u e Publishing
G oup; 2003.
9. Zhang W-W, Li L, Li D, Liu J, Li X, Li W, e al. The i s app o ed gene he apy
p oduc o cance Ad-p53 (Gendicine): 12 yea s in he clinic. Human gene he apy.
2018;29(2):160-79.
10. Bainb idge JW, Smi h AJ, Ba ke SS, Robbie S, Hende son R, Balaggan K, e
al. E ec o gene he apy on isual unc ion in Lebe 's congeni al amau osis. New
England Jou nal o Medicine. 2008;358(21):2231-9.
11. Ylä-He uala S. Endgame: glybe a inally ecommended o app o al as he
i s gene he apy d ug in he Eu opean union. Molecula The apy. 2012;20(10):1831-
2.
12. Shah ya i A, Saghaeian Jazi M, Mohammadi S, Raza i Nikoo H, Naza i Z,
Hosseini ES. De elopmen and Clinical T ansla ion o Gene The apy o Gene ic
Diso de s. F on ie s in gene ics. 2019;10:868.
13. B igh Focus Founda ion. Gene he apy o eye disease 2018, Oc oba 24
[A ailable om: h ps://www.b igh ocus.o g/macula /a icle/gene- he apy-eye-
disease.
14. Eskilsson E, Røsland GV, Solecki G, Wang Q, Ha e PN, G aziani G, e al.
EGFR he e ogenei y and implica ions o he apeu ic in e en ion in glioblas oma.
Neu o-oncology. 2017;20(6):743-52.
15. Alliance o egene a i e medicine. Selec Nea -Te m Clinical T ial Miles ones
& Da a Readou s: 2019+ 2019, Ma ch [A ailable om: h ps://alliance m.o g/.
16. genomecompile .
how o choose he pe ec ec o o you molecula biology expe imen 2016, Ma ch
21 [A ailable om: h p://www.genomecompile .com/how- o-choose- he-pe ec -
ec o /.
17. addgene. Plasmids 101: Wha is a plasmid? 2014, jan 14 [A ailable om:
h ps://blog.addgene.o g/plasmids-101-wha -is-a-plasmid.
18. Ha dee CL, A é alo-Soliz LM, Ho ns ein BD, Zechied ich L. Ad ances in non-
i al DNA ec o s o gene he apy. Genes. 2017;8(2):65.
19. Weile J, Hunzike J, Hall J. An i-miRNA oligonucleo ides (AMOs):
ammuni ion o a ge miRNAs implica ed in human disease? Gene he apy.
2006;13(6):496.
20. Pang X, Cui C, Wan S, Jiang Y, Zhang L, Xia L, e al. Bioapplica ions o cell-
SELEX-gene a ed ap ame s in cance diagnos ics, he apeu ics, he anos ics and
bioma ke disco e y: a comp ehensi e e iew. Cance s. 2018;10(2):47.
21. Ran FA, Hsu PD, W igh J, Aga wala V, Sco DA, Zhang F. Genome
enginee ing using he CRISPR-Cas9 sys em. Na u e p o ocols. 2013;8(11):2281.
22. Anzalone AV, Randolph PB, Da is JR, Sousa AA, Koblan LW, Le y JM, e al.
Sea ch-and- eplace genome edi ing wi hou double-s and b eaks o dono DNA.
Ta ge . 2019;5(3).
23. Lipocalyx. VIROMER® CRISPR FOR RNP DELIVERY 2019, Oc oba ,
[A ailable om: h ps:// i ome - ans ec ion.com/c isp .
24. Keele AM, Elmallah MK, Flo e TR. Gene he apy 2017: p og ess and u u e
di ec ions. Clinical and ansla ional science. 2017;10(4):242-8.
Chap e 2
36

Chap e 2
37
Gene he apy app oach aims o ea bo h inhe i ed and acqui ed diseases by
deli e ing a he apeu ic gene ic ma e ial o i s egula o y elemen s o a ge cells. The
u u e o gene he apy depends mainly on he success o design op imal ec o . Vi al
and non- i al ec o s ha e been used as gene deli e y ca ie s. In spi e o he ac ha
i al ec o s ha e he ad an age o high gene ans ec ion, he use o i al ec o s is
limi ed due o hei side e ec s. Thus, non- i al ec o s, ha e been de eloped and
applied in gene he apy o hei ad an ages, such as i s sa e y, high gene capaci y,
s abili y, chemical design flexibili y, and low immunogenic esponse. The e o e, he
main objec i e o his hesis o design no el niosome o mula ions, con aining no el
helpe lipid, able o deli e genes o eye and b ain, sa ely and e ec i ely.
1. To s udy he e ec o lycopene, as na u al helpe lipid, in niosome o mula ion
based on ca ionic lipid (DOTMA) and non-ionic su ac an (polyso ba e 60), o
boos ans ec ion e iciency in e inal pigmen epi helial (RPE-19) cells,
wi hou comp omising cell iabili y and using i o ans ec a e ina in i o.
2. To in es iga e he-lycopene con aining-niosomes o ans ec NT2 cells,
p ima y co ical cul u e as well as b ain co ex o a s, as sa e and e icien non-
i al ec o s o deli e DNA in o he CNS o ace many neu ological diso de s.
3. To ensu e he ole and impo ance o helpe molecule o u n on ans ec ion
e iciency. Niosomes o mula ion was p obed wi h di e en ca ionic lipid {2,3-
di ( e adecyloxy) p opan-1-amine (hyd ochlo ide sal )} and di e en helpe
molecule (chlo oquine diphospha e) o ans ec a e inal cells. he
inco po a ion o chlo oquine wi hin nano o mula ions, a he han as a co-
ea men o he cells, could open a new a enue o in i o e inal gene deli e y.
Chap e 2
38
Chap e 2
39
Chap e 3
Re inal gene deli e y
enhancemen by
lycopene inco po a ion
in o ca ionic niosomes
based on DOTMA and
polyso ba e 60
Chap e 3
38
Chap e 3
39
Re inal gene deli e y enhancemen by lycopene
inco po a ion in o ca ionic niosomes based on DOTMA and
polyso ba e 60
Mohamed Mashala,1, Noha A iaa,b,1, Gus a o Pu asa,c, Gema Ma ínez-Na a e ec,d, Edua do
Fe nándezc,d, Jose Luis Ped aza,c,
a NanoBioCel G oup, Labo a o y o Pha maceu ics, School o Pha macy, Uni e si y o he Basque Coun y
(UPV/EHU), Paseo de la Uni e sidad 7, 01006 Vi o ia-Gas eiz, Spain
b His ology and Cell Biology Depa men , Facul y o Medicine, Uni e si y o Alexand ia, Alexand ia, Egyp
c Ne wo king Resea ch Cen e o Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN), Vi o ia-
Gas eiz, Spain
d Neu op o hesis and Neu oenginee ing Resea ch G oup, Miguel He nández Uni e si y, Elche, Spain
Jou nal o Con olled Release 254 (2017) 55–64
ABSTRACT
The p esen s udy aimed o e alua e he inco po a ion o he na u al lipid
lycopene in o niosome o mula ions based on ca ionic lipid DOTMA and polyso ba e
60 non-ionic su ac an o analyze he po en ial applica ion o his no el o mula ion o
deli e gene ic ma e ial in o he a e ina. Bo h niosomes wi h and wi hou lycopene
we e p epa ed by he e e se phase e apo a ion me hod and physicochemically
cha ac e ized in e ms o size, ze a po en ial, polydispe si y index and capaci y o
condense, elease and p o ec he DNA agains enzyma ic diges ion. In i o
expe imen s we e pe o med in ARPE-19 cells a e complexion o niosomes wi h
pCMS-EGFP plasmid a app op ia e ca ionic lipid/DNA a ios. A 18/1 mass a io,
nioplexes con aining lycopene had nanome ic size, posi i e ze a po en ial, low
polydispe si y and we e able o condense, elease and p o ec DNA. Pe cen age o
ans ec ed cell was a ound 35% wi hou comp omising cell iabili y. The
in e naliza ion pa hways s udies e ealed a p e e ence o ca eolae media ed
endocy osis and mac opinocy osis, which could ci cum en lysosomal deg ada ion.
Bo h sub e inal and in a i eal adminis a ions o he a e ina showed ha nioplexes
we e able o ans ec efficien ly he ou e segmen s o he e ina, which offe
easonable hope o he ea men o many inhe i ed e inal diseases by a sa e non- i al
ec o o mula ion a e he less in asi e in a i eal adminis a ion.
Keywo ds: Niosomes, Lycopene, Gene he apy, Re ina, Non- i al ec o s,
Nano echnology

Chap e 3
40
3.1.In oduc ion
The abno mal exp ession o ac i i y o nume ous e inal p o eins has been
linked o he pa hogenesis o se e al blinding e inal diso de s wi h a gene ic
backg ound, such as Lebe congeni al amau osis [1], age- ela ed macula degene a ion
[2] o e ini is pigmen osa [3]. Un o una ely, mos o hese de as a ing condi ions do
no ha e effec i e ea men a he momen . Al hough no el app oaches, such as
enzyme/p o ein eplacemen and s em cell-based he apies ha e shown ecen ly
p omising esul s, gene he apy is by a he mos well-de eloped ield o esea ch o
he ea men o bo h inhe i ed and acqui ed e inal diso de s [4,5]. The unique
ana omical and his ological ea u es o he eye p o ide bo h bene i s and challenges o
he p og ess in gene-based ocula he apeu ics [6].
In he las decade, many i al and non- i al gene deli e y app oaches ha e been
de eloped o he ea men o many e inal pa hologies [7,8]. Compa ed wi h hei
coun e pa s, non- i al ec o s ha e a ac ed g ea a en ion as sa e al e na i e o
deli e gene ic ma e ial, since hey can ci cum en many sa e y issues ha a e s ill
associa ed wi h i al gene deli e y sys ems, such as immunogenici y, mu agenici y and
oncogenic effec s [9]. Consequen ly, he use o non- i al ec o s in clinical ials has
inc eased since 2004, while ha o i al ec o has dec eased signi ican ly [10].
Ac ually, ca ionic lipids and ca ionic polyme s a e he mos commonly used non- i al
ec o s [10,11]. Howe e , o da e, one o he main p oblems ha non- i al o mula ions
ha e o ace, in o de o each he clinical p ac ice, is hei limi ed ans ec ion
efficiency. The e o e, esea ch ac i i y on his a ea me i s special a en ion o he
scien i ic communi y [12].
As d ug deli e y sys em, niosomes ha e ecei ed g owing a en ion by ime o
being osmo ically ac i e and chemically s able o mula ions [13]. Besides, when i
comes o easy handling and low oxici y, hey a e conside ed qui e ad an ageous o e
he well-known liposomes [14].
Howe e , hei use as gene deli e y sys ems has been poo ly s udied, al hough some
ecen esul s ha e e ealed hei appealing p ope ies o ans ec efficien ly b ain and
e inal cells in a s [15–17].
Chap e 3
41
Niosomes, o gene deli e y pu poses, a e sel -assembled esicula nano ca ie
sys ems composed ypically by non-ionic su ac an , “helpe ” and ca ionic lipids [18].
The non-ionic “elec ically neu al” su ac an s enhance he s abili y o niosome
o mula ions [19]. Addi ionally, ca ionic lipids o m complexes by elec os a ic
in e ac ions upon he addi ion o nega i ely cha ged gene ic ma e ial [15,16], and
“helpe ” lipids ha e a ma ked in luence on bo h he physicochemical and biological
p ope ies o niosome gene ca ie s [15,17,20].
Recen ly, i has been epo ed on he li e a u e he la e ing p ope ies o he
“helpe ” lipid squalene (a na u al lipid ha belongs o he e penoid amily) in
ca ionic niosome gene deli e y o mula ions. The e o e, we decided o in es iga e he
effec ha lycopene, ano he na u al lipid, could ha e on a niosome o mula ion based
on ca ionic lipid N-[1-(2,3-dioleoyloxy)p opyl]-N,N,N- ime hylammonium chlo -ide
(DOTMA) and non-ionic su ac an polyso ba e 60.
Lycopene is a ca o enoid ha con ains 40 ca bon and 56 hyd ogen a oms (Fig.
1-C). Classically, i is known o be one o he mos po en na u al an ioxidan s ha
media e cy op o ec i e, immunomodula o y and an icance ac i i ies [21].
Fig. 1. Chemical s uc u es o he ca ionic lipid, N-[1-(2,3-dioleoyloxy)p opyl]-
N,N,N- ime hylammonium chlo ide (DOTMA) (A), Polyso ba e 60 (B), and
Lycopene (C).
Chap e 3
42
Addi ionally, lycopene can be ound a high concen a ion le els in he eye,
whe e i has shown bo h an i-in lamma o y and an i-angiogenic effec s [22].
We designed wo no el niosome ec o o mula ions o e inal gene deli e y
pu poses based on he same ca ionic lipid N-[1-(2,3-dioleoy-loxy)p opyl]-N,N,N-
ime hylammonium chlo ide (DOTMA) and he same non-ionic su ac an polyso ba e
60, in he absence/p esence o lycopene (DP60 and DP60L, espec i ely). Bo h
niosomes we e elabo- a ed by he sol en emulsi ica ion-e apo a ion echnique and
com-pa ed in e ms o pa icle size, polydispe si y index (PDI) and ze a po en ial. Upon
he addi ion o he epo ed pCMS-EGFP plasmid a diffe en ca ionic lipid/DNA a ios
(w/w), nioplexes we e ob ained and cha ac e ized by size, PDI, mo phology, and he
abili y o condense, elease and p o ec he DNA om enzyma ic diges ion. In i o
expe imen s we e pe o med o compa e he beha io o bo h ec o s in ARPE-19 cells
ega ding hei cellula up ake, ans ec ion efficiency, iabili y, and in e naliza ion
mechanism. Following he in i o cha ac e iza ion, he mos p omising o mula ion
was adminis e ed o a eyes ia in a i eal and sub e inal injec ion in o de o e alua e
ans ec ion efficiency by con ocal mic oscopy in bo h whole-moun and sagi al c oss
sec ions o he e ina.
3.2.Ma e ials and me hods
3.2.1. P oduc ion o ca ionic niosomes
Niosomes we e elabo a ed wi h sligh modi ica ions o he p e iously desc ibed
e e se phase e apo a ion me hod [23]. B ie ly, 5 mg o ca ionic lipid DOTMA D
(A an i Pola Lipids Inc., Alabama, USA) and 26 mg o polyso ba e 60 P60 (0.5%,
w/ , Sigma-Ald ich, Mad id, Spain) wi h/wi hou 1 mg lycopene L (Sigma-Ald ich,
Mad id, Spain) we e dissol ed in 1 ml o o ganic sol en , dichlo ome hane (Pan eac,
Ba celona, Spain). The emulsions we e ob ained by sonica ion o such o ganic phase
wi h 5 ml milliQ wa e o 30 s a 45 W (B anson Soni ie 250®, B anson Ul asonics
Co po a ion, Danbu y, USA). Dichlo o-me hane was emo ed om emulsions by
e apo a ion unde magne ic agi a ion o 2 h lea ing he ca ionic nanopa icles in he
aqueous medium. The co esponding mola a ios o bo h DP60 and DP60L
o mula ions we e 1:4 and 1:4:0.4, espec i ely.
Chap e 3
43
3.2.2.Plasmid p opaga ion and elabo a ion o nioplexes
pCMS-EGFP plasmid (5541 bp, Plasmid Fac o y, Biele eld, Ge many), was
p opaga ed in Esche ichia coli DH5-α and pu i ied using he Qiagen endo oxin- ee
plasmid pu i ica ion Maxi-p ep ki (Qiagen, Cali o nia, USA) acco ding o he
manu ac u e 's ins uc ions. The pu i ied plasmid DNA was quan i ied by measu ing
abso bance a 260 nm in a NanoD op® Spec opho ome e (The mo Fishe Scien i ic
Inc. Den e , USA). The pu i y o he plasmid was e i ied by aga ose gel
elec opho esis (Bio-Rad, Mad id, Spain) in T is Bo a e-EDTA buffe , pH 8.0 (TBE
buffe ). DNA bands we e de ec ed using GelRed™ (Bio-Rad, Mad id, Spain) o s ain
DNA, and images we e obse ed wi h a ChemiDoc™ MP Imaging Sys em (Bio-Rad,
Mad id, Spain). The s ock solu ion o pCMS-EGFP plasmid (0.5 mg/ml) was es ima ed
o be a ound 0.14 μM.
Bo h DP60 and DP60L nioplexes (niosome/DNA complexes) we e elabo a ed
by mixing an app op ia e olume o a s ock solu ion o pCMS-EGFP plasmid (0.5
mg/ml) wi h diffe en olumes o he niosome suspensions (1 mg ca ionic lipid/ml) o
ob ain diffe en ca ionic lipid/DNA mass a ios (w/w). The mix u e was le o 30 min
a oom empe a u e o enhance elec os a ic in e ac ion be ween he ca ionic niosomes
and he nega i ely cha ged plasmid.
3.2.3.Cha ac e iza ion o niosomes/nioplexes
Pa icle size and polydispe si y index (PDI) we e de e mined by dynamic ligh
sca e ing (DLS) wi h Ze asize Nano ZS (Mal e n Ins umen s, UK). De e mina ion
o ze a po en ial by Lase Dopple
Velocime y (LDV) was ca ied ou wi h he same ins umen , whe e samples we e
dispe sed in a 0.1 mM NaCl solu ion. Pa icle size, epo ed as hyd odynamic
diame e , was ob ained by cumula i e analysis. All measu emen s we e ca ied ou in
iplica e.
Chap e 3
50
eagen Lipo ec amine™2000 signi ican ly dec eased o 82% (p < 0.05). The
mic og aphs ob ained in (Fig. 3B) demons a ed ha ans ec ed ARPE-19 cells
main ained a no mal mo phology wi h bo h nioplexes e en wi h high ca ionic
lipid/DNA mass a ios.
Fig. 2. Physicochemical cha ac e iza ion o nioplexes. A) Effec o ca ionic lipid/DNA
mass a io (w/w) on bo h pa icle size (ba s) and ze a po en ial (lines). Each da a poin
ep esen s he mean ± SD (n = 3). TEM o DP60 (B1) and DP60L nioplexes (B2) a
a io o 18/1 ca ionic lipid/DNA mass a io (w/w). Scale ba = 500 nm. Binding, SDS-
induced elease and p o ec ion o DNA a diffe en ca ionic lipid/DNA mass a ios
(w/w) o nioplexes based on bo h DP60 (C1) and DP60L (C2) isualized by aga ose
elec opho esis. Lanes 1–3 co espond o uncomplexed DNA; lanes 4–6, ca ionic
lipid/DNA mass a io 6/1; lanes 7–9, ca ionic lipid/DNA mass a io 12/1; lanes 10–12,
ca ionic lipid/DNA mass a io 18/1; lanes 13–15, ca ionic lipid/DNA mass a io 22/1.
Nioplexes we e ea ed wi h SDS (lanes 2, 5, 8, 11 and 14) and DNase I + SDS (lanes
3, 6, 9, 12 and 15). OC: open ci cula o m, SC: supe coiled o m.
3.3.3.Cell up ake s udies

Chap e 3
51
Nioplexes a ca ionic lipid/DNA mass a io o bes ans ec ion, 18/ 1, we e used
o de e mine up ake pe cen age in ARPE-19 cells. Fig. 4-A ea u es he pe cen age o
FITC-posi i e cells quan i ied by low cy ome y. In gene al, he up ake pe cen age
inc eased o e ime o bo h o mula ions. Addi ionally, DP60 up ake alues (50.5%,
62.4%; and 75.5%) we e signi ican ly highe (p < 0.05) han alues o DP60L up ake
(5.8%, 11.1% and 23%) a he ime poin s es ed, espec i ely.
Fig. 3. In i o ans ec ion efficiency and cell iabili y in ARPE-19 cells a 72 h pos -
ans ec ion. (A) Flow cy ome y-based e alua ion o he pe cen age o EGFP-posi i e
cells (ba s) and pe cen age o iable cells (lines) a diffe en ca ionic lipid/DNA mass
a ios (w/w). Values ep esen mean ± SD (n = 3). (*P < 0.05 s. Lipo ec amine™2000
ans ec ion). (#P < 0.05 s. Lipo ec amine™2000 iabili y). (B) O e lay o
luo escence and phase-con as mic og aphs o ARPE-19 cells 72 h pos - ans ec ion
a diffe en ca ionic lipid/DNA mass a ios (w/w). Scale ba = 100 μm.
Con ocal mic og aphs we e ob ained a e 2 and 4 h o incuba ion o isualize
he p og ess o complex in e naliza ion o e ime. Fig. 4-B showed ha bo h nioplexes
we e homogeneously dis ibu ed wi hin he cy oplasm 2 h o incuba ion. Howe e , a e
4 h o incuba ion, cy oplasmic agg ega es o DP60 complexes we e disce ned compa ed
o hei DP60L coun e pa s ha main ained hei ela i e homogeneous cy oplasmic
dis ibu ion.
Chap e 3
52
Fig. 4. Up ake o FITC-labeled nioplexes in ARPE-19 cells. Bo h DP60 and DP60L a
a mass a io o 18/1 (w/w). (A) Pe cen age o FITC-posi i e cells. Da a ep esen mean
± SD (n = 3). *p < 0.05. (B) Fluo escence mic og aphs o ARPE-19 cells a 2 h and 4
h o incuba ion wi h FITC-labeled DP60 and DP60L nioplexes (g een). Nuclei s ained
wi h Dapi (blue). O iginal magni ica ion 63×. Scale ba = 20 μm. (Fo in e -p e a ion
o he e e ences o colo in his igu e legend, he eade is e e ed o he web e sion
o his a icle.)
3.3.4. Cell in e naliza ion s udies
Fig. 5 illus a es ha DP60 nioplexes (g een) highly co-localized wi h each one
o he h ee diffe en endocy osis ma ke s used ( ed), esul ing in yellowish signals ha
we e quan i ied by Mande 's o e lap coefficien . Values o M ≥ 0.6 indica ed a posi i e
co-localiza ion. Howe e , DP60L complexes, co-localized mainly wi h chole a oxin
(M = 0.76 ± 0.04) and dex an (M = 0.76 ± 0.07) endocy osis ma ke s, whe eas no
posi i e co-localiza ion was obse ed wi h ans e in (M = 0.53 ± 0.03).
Chap e 3
53
Fig. 5. Th ee-channel o e lay RGB images o ARPE-19 cells showing nioplexes wi h
FITC-labeled pCMS-EGFP (g een) and one o he endocy osis ma ke s in ed
(AlexaFluo ® 555-Chole a Toxin, AlexaFluo ® 546-T ans e in o AlexaFluo ® 594-
dex an). P esence o yellow/o ange colo ep esen s he o e lay o an endocy ic ma ke
and nioplexes. (M = Mande 's o e lap coefficien ). O iginal magni ica ion 63×, Scale
ba = 25 μm. (Fo in e p e a ion o he e e ences o colo in his igu e legend, he
eade is e e ed o he web e sion o his a icle.)
3.3.5.His ology and immuno luo escence analysis o EGFP exp ession in i o
A e 72 h o in a i eal adminis a ion o DP60L nioplexes, EGFP exp ession in
he a e ina was analyzed by CLSM (Fig. 6). The analysis o whole-moun
p epa a ions (Fig. 6-A, B) e ealed na i e EGFP-exp ession in some ganglion cells
con aining NeuN immuno eac i i y ( ed colo ) as well as in some cells in con ac wi h
a ypical mic oglial-like mo phology, while no luo escence was de ec ed in con ol
e ina (da a no shown). Fig. 7 shows e ical e inal sec ions whe e EGFP exp ession
was de ec ed in bo h ganglion cell laye (GCL) labeled wi h an i-NeuN ( ed colo ), and
ou e segmen s (OS) o pho o ecep o s wi h an i-Reco e in, a ma ke o pho o ecep o s
Chap e 3
54
( ed colo ) a e in a i eal (Fig. 7-A, B) and sub e inal injec ion (Fig. 7-C, D),
ega dless he ou e o adminis a ion. Bo h posi i e con ols o a e inae ans ec ed
wi h Lipo ec amine™ 2000 and nega i e con ols o non- ans ec ed e inae can be
obse ed in he Supplemen a y da a sec ion (Fig. S1).
3.4.Discussion
Due o i s appealing chemical s uc u e, he comme cially a ailable ca ionic lipid
DOTMA has been widely used o gene deli e y applica ions [25]. As shown in Fig. 1-
A, i s s uc u e is composed o a pola head-g oup, wo non-pola hyd ophobic chains,
a linke and a back-bone, which classically a e known as he ou domains ha ule
gene ans ec ion p ocess [26].
We combined DOTMA wi h he non-ionic su ac an polyso ba e 60, in a niosome
o mula ion a a mola a io o 1:4 espec i ely, in o de o enhance cell ole ance [27]
and p o ide a s e ic ba ie o a oid agg ega ion [28]. I has been epo ed on he
li e a u e ha he p esence o PEG chains in he chemical s uc u e o polyso ba es (Fig.
1-B) p o ides physicochemical s abili y o lipid o mula ions [29], conse es
effec i eness o e ime and boos s ans ec ion efficiency [30]. Compa ed wi h
polyso ba e 80, ano he polyso ba e ha has been widely used in he elabo a ion o
niosome o mula ions o gene deli e y applica ions, [15–17,31] polyso ba e 60 could
offe some impo an ad ances. Fo ins ance, he lack o double bonds in he
hyd oca bon chains (Fig. 1-B) could p o ide low pe meabili y o he esicles, and
he e o e be e s abili y o niosome memb anes [29]. Addi ionally, compa ed wi h
o he hyd ophilic su ac an s such as polyso ba es 80, 40 o 20, he low hyd ophilic-
lipophilic balance (HLB) alue o polyso ba e 60 (14.9) could help o solubilize
lycopene mo e efficien ly [31]. The addi ion o he na u al and non-pola lipid lycopene
(Fig. 1-C) in o niosome bilamina memb ane could inc ease i s luidi y, dis u b
memb ane packing, and consequen ly esicle suscep ibili y o en i onmen al s esses
[32].
Chap e 3
55
Fig. 6. Immunohis ochemical s udy o EGFP exp ession in e inal whole-moun
p epa a ions 3 days a e in a i eal adminis a ion o DP60L nioplexes. Pa ial
colocaliza ion o EGFP (g een colo ) wi h NeuN-posi i e ganglion cells ( ed) was
obse ed in he ganglion cell laye (GCL) ( ed). EGFP exp ession was obse ed as well
in some cells wi h ypical mic oglial mo phology in GCL (NeuN-nega i e cells). Nuclei
we e s ained wi h Hoechs 33342 (blue). Scale ba s: 10 μm. (Fo in e p e a ion o he
e e ences o colo in his igu e legend, he eade is e e ed o he web e sion o his
a icle.)
Once elabo a ed by he e e se phase elabo a ion me hod, bo h DP60 and DP60L
niosomes showed app op ia e size (in he nanome ic scale) and PDI alues (below 0.5)
o gene deli e y pu poses (Table 1). High posi i e ZP alues (> +25 mV) ensu e long-
las ing s abili y [33], spon aneous elec os a ic in e ac ion wi h DNA, as well as binding
o he esul ing nioplexes o he nega i ely cha ged componen s o he cell memb ane
p io o cellula up ake [26].
To elabo a e nioplexes, we added pCMS-EGFP epo e plasmid o bo h niosome
o mula ions a diffe en ca ionic lipid/DNA mass a ios, since o he wise, he complex
assembly p ocess could be slowed down [34]. The sligh changes disce ned in he size
o nioplexes (100–150 nm, Fig. 2-A), a he mass a ios s udied, migh be due o he
delica e balance o diffe en e en s in ol ed in he mul is ep sel -assembled complex
o ma ion, such as: elec os a ic in e ac ion, u he memb ane me ging, lipid mixing

Chap e 3
56
and agg ega e g ow h [34]. Rega ding he ZP alues, he g adual inc ease o supe icial
cha ge along wi h ca ionic lipid/DNA a ios (w/w) sugges s he capaci y o ca ionic
niosomes o bind o and neu alize he nega i ely cha ged phospha e g oups o DNA
[17]. Lycopene addi ion educed ZP alue o DP60L nioplexes, compa ed o DP60, a
all a ios s udied. This ac could be explained by he pe u ba ion o he lipid memb ane
bilaye , which could dissipa e he elec ical po en ial (Fig. 2-A) [35]. In any case, bo h
o mula ions could unc ion as gene deli e y ca ie s, since he posi i ely cha ged
complexes could in e ac elec os a ically wi h he anionic cell coa , inducing ea ly
s eps o he endocy osis p ocess [36]. The high posi i e ZP alue o bo h DP60 and
DP60L nioplexes, especially a 18/1 mass a io (42 and 27 mV, espec i ely), could
ensu e he disc e e mo phology and absence o agg ega es obse ed by TEM
mic og aphs (Fig. 2-B) [[37].].
Among o he ac o s ha can in luence on he ans ec ion p ocess, he
elec os a ic in e ac ions be ween he nega i ely cha ged phospha e g oups o he DNA
and he posi i ely cha ged amine g oups o he ca ionic niosomes me i s special
a en ion [15–17,38]. We obse ed by aga ose gel elec opho esis assay ha a all
ca ionic lipid/DNA a ios es ed, bo h niosomes we e able o condense, elease and
p o ec he DNA om enzyma ic diges ion (Fig. 2-C1and C2).
Once we e alua ed ha ou o mula ions we e bio echnologically sui able o
gene deli e y pu poses, we p oceeded o e alua e hei biological pe o mance in
ARPE-19 cells.
ARPE-19 cell line has a no mal ka yo ype and has unc ional and s uc u al p ope ies
simila o e inal pigmen epi helia (RPE) in i o, exp esses RPE-speci ic ma ke s,
hence i is conside ed a sui able ans ec ion model o in es iga e ou ec o s'
effec i eness and sa e y be o e i s applica ion in i o [39]. I has been epo ed ha he
non-ionic na u e o su ac an s makes niosomes well ole a ed by cells [40]. Ou esul s
in Fig. 3 show highe cell iabili y alues in cells ans ec ed wi h bo h nioplexes when
compa ed wi h cells ans ec ed wi h Lipo ec amine™ 2000. Addi ionally, we obse ed
unde he luo escence mic oscope ha cells ans ec ed wi h bo h nioplexes
main ained hei no mal mo phology, e en a high ca ionic lipid/DNA a ios (Fig. 3-
Chap e 3
57
B). Al hough he pe cen age o ans ec ed cells wi h DP60L niosomes a 18/1 mass
a io was signi ican ly lowe han ha ob ained wi h comme cially a ailable
Lipo ec amine™ 2000, ou niosomes o mula ion was be e ole a ed by ARPE-19
cells. The e o e, i could be an in e es ing al e na i e o Lipo ec amine™ 2000, since
some au ho s ha e epo ed damage on he e ina associa ed o he in i o
adminis a ion o Lipo ec amine™ 2000 in he eye [41]. Rega ding he ans ec ion
efficiency, he lipid composi ion is conside ed a p ima y limi ing ac o ha affec s o
his p ocess [25]. We clea ly obse ed in Fig. 3 he impac ha lycopene had on
ans ec ion efficiency in ARPE-19 cells, since alues we e clea ly highe when
lycopene was p esen in he niosome o mula ion. Al hough he exac mechanism o
lycopene ac ion has no ye been ully elucida ed, some au ho s sugges he exis ence
o a lycopene ecep o and/o anspo e in he nuclea memb ane o cells [42].
Addi ionally, o he s udy has documen ed he capaci y o lycopene o modula e
ansc ip ion [43]. Such effec could be ei he by di ec in e ac ions wi h ansc ip ion
ac o s such as nuclea ac o -kappa, o by indi ec modi ica ions o ansc ip ional
ac i i y. In any case, u he esea ch is s ill needed o de e mine he exac mechanism
[44]. The ascending ans ec ion pe cen ages ob ained by DP60L a high mass a ios
migh be a ibu ed, pa ially, o he igge ing effec o ee niosomes abso bed on o
he cell memb ane. A highe ca ionic lipid/DNA mass a ios, he e is a la ge excess o
ca ionic lipid o DNA, he e o e a popula ion o ee niosomes is expec ed [45]. This
ee ca ionic lipid could p olong cellula e en ion o dec eases deg ada ion a e o DNA
[25]. None heless, addi ional expe imen s a e needed o elucida e he de ailed
mechanisms in ol ed.
Chap e 3
58
Fig. 7. Con ocal luo escence mic og aphs o e inal c oss-sec ions a e 3 days o
in a i eal (A,B) and sub e inal (C,D) adminis a ion o DP60L nioplexes. A e
in a i eal injec ions, EGFP luo escence was obse ed in glial cells in he GCL, and
in he ou e segmen s o he pho o ecep o s. Localiza ion o EGFP a e sub e inal
injec ions was de ec ed as well in OS o pho o ecep o s (s ained wi h eco e in, in ed)
and some mic oglial cells. Cell nuclei we e coun e s ained wi h Hoechs 33342 (blue).
Scale ba s: 20 μm. (Fo in e p e a ion o he e e ences o colo in his igu e legend,
he eade is e e ed o he web e sion o his a icle.)
Chap e 3
59
To de e mine whe he enhanced in e naliza ion was among he effec s ha lycopene
inco po a ion could ha e in niosome o mula ions, we s udied he pe cen age o cellula
up ake o bo h DP60 and DP60L o mula ions a he mass a io o bes ans ec ion
efficiency (18/1) in ARPE-19 cells a diffe en imes (Fig. 4). Su p isingly, low
cy ome y s udies showed ha lycopene addi ion clea ly educed he pe cen age o
cellula up ake a all imes s udies when compa ed o DP60 o mula ion (Fig. 4-A).
Such educ ion in he cellula up ake could p obably be due o he lowe ze a po en ial
o DP60L o mula ion compa ed wi h DP60 (Fig. 2-A).
Addi ionally, CLSM s udied (Fig. 4-B), excluded me e elec os a ic adhe ence o
ca ionic nioplexes o he nega i ely cha ged su ace o ARPE-19 cell, since a clea
in acellula dis ibu ion o bo h nioplexes was obse ed in he case o bo h
o mula ions. In any case, he cy oplasma ic dis ibu ion o bo h nioplexes showed a
diffe en beha io . Whe eas DP60L nioplexes main ained a homogeneous dis ibu ion
in he cy oplasm o e he ime, DP60 nioplexes showed some agg ega es a 4 h. The
diffe ences obse ed in he cy oplasma ic dis ibu ion o bo h o mula ions could
sugges diffe en in e naliza ion pa hways. The e o e, and mo i a ed by he diffe ences
obse ed be ween bo h o mula ion in e ms o ans ec ion efficiency and cellula
up ake, we nex s udied he cellula afficking o bo h nioplexes a he mass a io o
bes ans ec ion efficiency (18/1). Th ee o he pa hways mos employed in he up ake
p ocesses we e assayed; cla h in-media ed endocy osis (CME), ca eola-media ed
endocy osis (C ME) and mac opinocy osis (Fig. 5) [9]. The pe o mance o non- i al
ec o s is known o be clea ly affec ed by hei dis inc cellula in e naliza ion pa hway,
aking in o accoun he a iable effec i eness o e e y pa hway in he elease o DNA
in o he cy oplasm, which is one o he c i ical s eps in he e en ual ansgene
exp ession [34]. Al hough he e is no a clea consensus in he scien i ic communi y, i
is widely accep ed ha he endolysosomal a e is he hallma k ea u e o CME [37,46].
In he o he hand, C ME and mac opinocy osis a e widely ela ed o non-acidic and
non-diges i e ou es o cellula up ake [47,48]. The e o e, ou esul s obse ed in Fig.
5 sugges ha in e naliza ion o DP60L ia hese las pa hways could be ad an ageous
Chap e 3
66
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Chap e 3
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Chap e 4
69
Chap e 4
Non- i al ec o s
based on ca ionic
niosomes as e icien
gene deli e y
ehicles o cen al
ne ous sys em cells
in o he b ain
Chap e 4
70
Chap e 4
71
Non- i al ec o s based on ca ionic niosomes as e icien gene deli e y
ehicles o cen al ne ous sys em cells in o he b ain
Mohamed Mashala,1, Noha A iaa,b,1, C is ina So o-Sánchezc,d, Gema Ma ínez-Na a e ec,d,
Edua do Fe nándezc,d, Gus a o Pu asa,c,⁎, José Luis Ped aza,c,⁎
a NanoBioCel G oup, Labo a o y o Pha maceu ics, School o Pha macy, Uni e si y o he Basque Coun y
(UPV/EHU), Paseo de la Uni e sidad 7, 01006 Vi o ia-Gas eiz, Spain b His ology and Cell Biology Depa men ,
Facul y o Medicine, Uni e si y o Alexand ia, Alexand ia, Egyp c Biomedical Resea ch Ne wo king Cen e in
Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN), Spain d Neu op o hesis and Neu oenginee ing
Resea ch G oup, Miguel He nández Uni e si y, Elche, Spain
Abs ac
De elopmen o sa e and e icien non- i al ec o s o deli e DNA in o he CNS
ep esen s a huge challenge o ace many neu ological diso de s. We elabo a ed
niosomes based on DOTMA ca ionic lipid, lycopene “helpe ” lipid and polyso ba e 60
as non-ionic su ac an s o gene deli e y o he CNS. Niosomes, and hei
co esponding nioplexes ob ained a e he addi ion o he pCMS-EGFP plasmid, we e
cha ac e ized in e ms o size, cha ge, mo phology and capaci y o condense, elease
and p o ec DNA. In i o expe imen s we e pe o med in NT2 cells o e alua e
ans ec ion e iciency, iabili y, cellula up ake and in acellula dis ibu ion.
Addi ionally, ans ec ion in p ima y co ex cells we e pe o med p io o b ain
adminis a ion in o a ce eb al co ex. Da a ob ained showed ha nioplexes exhibi ed
no only adequa e physicochemical p ope ies o gene deli e y applica ions, bu also
ele an ans ec ion e iciencies (17%), wi hou hampe ing iabili y (90%).
In e es ingly, In i o expe imen s depic ed p omising p o ein exp ession in bo h
co ical glial cells and blood essels.
Keywo ds: Cen al ne ous sys em, Gene he apy, Non- i al ec o s, Niosomes,
Ca ionic lipids

Chap e 4
72
4.1.In oduc ion
Gene he apy concep elies on he in oduc ion o gene ic ma e ial in o a ge cells o
modi y p o ein exp ession o he apeu ic pu poses (Pezzoli e al., 2012). O e he ime
has eme ged as a p omising s a egy o he ea men o many diseases. F om a p ac ical
poin o iew, he ne ous sys em ans ec ion ep esen s a huge challenge o add ess
many de as a ing neu ological diso de s such as Pa kinson o Alzheime diseases
(Nob e and Almeida, 2011) ha a e di icul o ea wi h adi ional pha macology
app oaches, mainly due o he b ain physical ba ie s ha d ugs need o o e come a e
sys emic adminis a ion and he complexi y o he sys em (Nagabhushan Kalbu gi e
al., 2013). Howe e , nowadays, gene he apy clinical ials o neu ological diso de s
a e s ill ew in numbe , mainly due o he lack o sa e and sui able app oaches o deli e
gene ic ma e ial o a ge s cells (Yin e al., 2014). Non- i al ec o s ha e ecei ed
inc easing a en ion hanks o hei la e ing p ope ies such as; easy elabo a ion, low
cy o oxici y, immune ole ance and lack o oncogenic e ec s. Addi ionally, i is
possible o use la ge DNA inse s (Mansou i e al., 2004; Tang and Szoka, 1997).
The e o e, a no able shi o p eclinical s udies has occu ed om i al o non- i al
ec o s in a ious applica ions, including neu ological diso de s (Pelu o e al., 2015).
Howe e , he exp ession o genes deli e ed ia such ec o s is ypically low, which
jus i ies he need o u he esea ch in his opic. Among non- i al ec o s, niosomes
ha e ecen ly eme ged as p omising gene deli e y sys ems (A ia e al., 2017; Ochoa e
al., 2014; Ojeda e al., 2016c; Pu as e al. 2014, 2015). Basically, niosomes a e non-
phospholipid liposome like esicles, wi h a bilaye s uc u e (Ojeda e al., 2016a).
Compa ed o liposomes, niosomes a e ecognized o hei low cos and supe io
chemical and s o age s abili ies (Raje a e al., 2011). The ypical componen s o a
niosome o mula ion include he ca ionic lipid, which elec os a ically in e ac s wi h
nega i ely cha ged gene ic ma e ial o o m complexes, known as nioplexes (Agi e e
al., 2015). The “helpe ” lipid, which enhances physicochemical p ope ies o he
o mula ion and he in acellula disposi ion o he complexes (Ojeda e al., 2016b), and
he non-ionic su ac an s, which inc ease he s abili y o he o mula ion and a oid
agg ega ion be ween esicles (Huang e al., 2011). Among ca ionic lipids, DOTMA has
been widely used o elabo a e lipid nanopa icles o gene deli e y applica ions, mainly
Chap e 4
73
due o i s high aqueous solubili y and i s appealing chemical s uc u e, which includes
a pola head-g oup, a linke , a backbone and a hyd ophobic domain (Rezaee e al.,
2016). Lycopene is a na u al ca o enoid known o i s po en an ioxidan p ope ies
(K ishnamoo hy e al., 2013). Recen ly, has been used as “helpe ” lipid o enhance
ans ec ion e iciency in e ina (Mashal e al., 2017). Polyso ba es con ain polye hylene
glycol (PEG) chains ha imp o e ans ec ion e iciency o liposome o mula ions
(Meye e al., 1998). The e o e, polyso ba es a e classically inco po a ed in o niosome
o gene deli e y applica ions as non-ionic su ac an s.
4.2.Ma e ial and me hods
4.2.1. P epa a ion o niosomes and nioplexes
The e e se phase emulsi ica ion e apo a ion echnique was used o
elabo a ion o niosomes as p e iously desc ibed (Mashal e al., 2017). B ie ly, he
ca ionic lipid DOTMA (0.1% w/ , A an i Pola Lipids Inc. Alabama, USA), non-ionic
su ac an Polyso ba e 60 (0.5% w/ , Sigma Ald ich, Mad id, Spain) and helpe lipid
Lycopene (0.02% w/ , Sigma Ald ich Mad id, Spain) we e mixed a a mola a io o
1/4/0.4 and dissol ed in 1 ml o he o ganic sol en dichlo ome hane (Pan eac,
Ba celona, Spain). The chemical s uc u e o such componen s can be obse ed on Fig.
1. Nex , 5 ml miliQ wa e we e added and he emulsion was sonica ed o 30 s a 45 W
(B anson Soni ie 250, Danbu y, USA). A e 2 h o magne ic e apo a ion, he o ganic
phase was emo ed, lea ing DP60L ca ionic niosomes suspended in he aqueous phase
a a 1 mg/ml ca ionic lipid DOTMA concen a ion. Nioplexes we e o med by mixing
a s ock solu ion o pCMS-EGFP plasmid (0.5 mg/ml) wi h niosome suspension. Unde
gen le pipe ing, di e en ca ionic lipid/DNA (w/w) a ios we e elabo a ed. The mix u e
was le o 30 min a oom empe a u e.
4.2.2. Size and ze a po en ial measu emen
The hyd odynamic diame e and ze a po en ial o bo h niosomes and nioplexes,
we e measu ed by Dynamic Ligh Sca e ing (DLS) and Lase Dopple Velocime y
(LDV) using Ze asize Nano ZS (Mal e n Ins umen , UK). Pa icle size epo ed as
Chap e 4
74
hyd odynamic diame e was ob ained by Z-a e age. All measu emen s we e ca ied ou
in iplica es. Only da a ha me he quali y c i e ia we e included in he s udy.
4.2.3. C yo-TEM analysis
The mo phology o o mula ions was e ealed by C yo-TEM analysis and
samples we e examined by a TEM, TECNAI G2 20 TWIN (FEI), ope a ing a an
accele a ing ol age o 200 KeV in a b igh - ield and low-dose image mode (Ojeda e
al., 2015). Digi al images we e acqui ed wi h digi al came a.
Fig. 1. Chemical composi ion o DP60L ca ionic niosomes. (A) Ca ionic lipid
DOTMA-Cl, (B) Polyso ba e 60 and (C) Lycopene.
4.2.4. Aga ose gel elec opho esis s udies
The abili y o he niosomes o condense, elease and p o ec plasmid DNA
agains enzyma ic diges ion was assayed by aga ose gel elec opho esis. Nioplexes
we e analyzed a di e en ca ionic lipid/DNA (w/ w) a ios (200 ng o DNA/well). The
aga ose gel (0.8%, w/ ) was imme sed in a T is-ace a e-EDTA bu e and exposed o
30 min o 120 V. DNA bands we e s ained wi h GelRed® and obse ed wi h a
ChemiDoc® MP Imaging Sys em. SDS solu ion (3%) and DNase I enzyme (1 U DNase
I/2.5 µg DNA) we e added o he samples o e alua e he elease and p o ec ion,
Chap e 4
75
espec i ely. The in eg i y o DNA in each sample was compa ed o a con ol o
un ea ed DNA.
4.2.5. NT2 cell cul u e and in i o ans ec ion
NT2 cells (ATCC®−CRL,1973) we e cul i a ed in comple e medium,
Dulbecco’s Modi ied Eagle’s Medium (DMEM), en iched wi h 10% e al bo ine se um
(FBS) and an ibio ics (100 U/ml penicillin and 100 μg/ml s ep omycin, Gibco®, Li e
Technologies S.A., Mad id, Spain). Be o e ans ec ion, NT2 cells we e seeded in 24-
well pla es a an ini ial densi y o 8 ×104 cells/well and allowed o g ow o 70–80%
con luence. Then, he medium was eplaced wi h se um- ee Op i-MEM (Gibco®, Li e
Technologies S.A., Mad id, Spain), and cells we e exposed o nioplexes (1.25 μg o
pCMS-EGFP/well). A e 4 h o incuba ion, ans ec ion medium was emo ed and
e eshed wi h comple e medium. Cells we e allowed o g ow o 24 h un il being
analyzed by low cy ome y (FACSCalibu , BD, San Jose, USA). Posi i e con ol
(Lipo ec amine® 2000, Gibco®, Li e Technologies S.A., Mad id, Spain) was p epa ed
ollowing he manu ac u e ’s p o ocol.
4.2.6. Cellula up ake and in acellula dis ibu ion s udies
NT2 cells we e cul u ed as p e iously men ioned. The egula g ow h media
was emo ed and cells we e exposed o nioplexes (p epa ed wi h FITC-labeled pCMS-
EGFP plasmids, Da eBio. Mad id, Spain). A e 4 h o incuba ion, he ans ec ion
medium was emo ed and cells we e washed wi h PBS, ypsinized and analyzed by
FACSCalibu low cy ome e . 10.000 e en s we e collec ed and analyzed o each
sample. Each sample was analyzed in iplica e.
To de e mine he in acellula dis ibu ion o in e nalized nioplexes, cells we e seeded
on co e slips (24-well pla es) and exposed o he nioplexes. The in acellula
dis ibu ion o nioplexes was analyzed by con ocal lase scanning mic oscopy (CLSM;
Olympus Fluo iew 500).
4.2.7. Endocy osis mechanism
Chap e 4
82
Fig. 4. Cellula up ake s udies in NT2 cells 4 h pos incuba ion. (A) Flow cy ome y
do -plo s (SSC-H and FL1) o con ol cells wi hou ea men (A1), naked DNA (A2),
DP60L nioplexes a 14/1 (w/w) a io (A3), and lipo ec amine 2000® (L2K) a 2/1(w/w)
a io (A4). (B) Flow cy ome y measu emen o NT2 cells ea ed wi h FITC-labeled
o mula ions. E o ba s ep esen SD (n = 3). *p < 0.05. (C) Fluo escence mic oscopy
images o NT2 cells a e 4 h o incuba ion wi h FITC-labeled DP60L nioplexes a 14/1
(w/w) a io. Cells we e s ained wi h DAPI- luo omoun G (blue). Whi e a ows indica e
nanopa icles a ound he nucleus. (Scale ba =25 μm). (Fo in e p e a ion o he
e e ences o colou in his igu e legend, he eade is e e ed o he web e sion o
his a icle.)

Chap e 4
83
Fig. 5. In e naliza ion s udies. (A) Con ocal mic oscopy images showing in acellula
dis ibu ion in NT2 cells o nioplexes a 14/1 a io labelled wi h FITC-pCMS EGFP
(g een) plasmid and AlexaFluo 555-Chole a Toxin (A1), AlexaFluo 546-T ans e in
(A2), AlexaFluo 594-Dex an (A3), and LysoT acke Red DND-99 ma ke s (A4), all
in ed. (M1 = Mandeŕs o e lap coe icien ). Scale ba s =25 µm. (B) E ec o endocy ic
inhibi o s on he cellula up ake o FITC-labeled nioplexes. Da a we e no malized o
up ake wi hou inhibi o . (C) Aga ose gel elec opho esis DNA eleased p o iles o
lipoplexes and nioplexes incuba ed wi h o wi hou PS. (D) Quan i ica ion o eleased
DNA in aga ose gel elec opho esis.
men ioned endocy osis mechanisms we e simul aneously inhibi ed by p io ea men
wi h me hyl cyclodex in (28%). Howe e , MPC inhibi ion by wo mannin, as an
inhibi o o MPC, had less e ec on cellula up ake educ ion, since up ake alues
whe e highe , a ound 80%. The capaci y o DNA compac ed wi h bo h DP60L
niosomes and lipo ec amine® 2000 o escape om he la e endosome, simula ed by PS
micelles (167 nm, and −65 mV), was analyzed by aga ose gel elec opho esis assay
(Fig. 5-C). As no iced in Fig. 5-D, in he p esence o PS micelles, he amoun o DNA
eleased om lipoplexes was a ound 20%, while in he case o nioplexes,
app oxima ely, double amoun o DNA, 40%, was eleased. Wi hou he p e ious
incuba ion wi h he anionic PS micelles, he eleased DNA was almos 0% in he case
Chap e 4
84
o lipoplexes, and abou 30% wi h DP60L nioplexes. Addi ionally, he pH i a ion
cu e (Supplemen a y da a, Fig. 1) p o ided e idence o he bu e ing capaci y o
DP60L o mula ion, since pH alue, sligh ly dec eased om 4.1 o 1.7, a e he
addi ion o 1.400 µL o HCl 0.1 M.
4.1.1. P ima y co ical neu on and in i o gene exp ession s udies
Fig. 6. P ima y co ical cul u e and in i o gene exp ession o EGFP ca ied by DP60
nioplexes a 14/1 mass a io (w/w). A1 and A2, ans ec ion o p ima y neu onal cell
cul u es 24 h pos ans ec ion. NeuN-posi i e neu ons ( ed) and nuclei coun e s ained
wi h Hoechs 33342 (blue) (Scale ba s =20 and 40 μm, espec i ely). B, in i o gene
exp ession o pCMS-EGFP 72 h a e in aco ical adminis a ion o nioplexes. Nuclei
a e shown in blue (Hoechs ), neu ons in ed (NeuN+), and EGFP exp ession (GFP+) in
g een. Whi e a ow poin s a blood essel. Scale ba 40 μm. (Fo in e p e a ion o he
e e ences o colou in his igu e legend, he eade is e e ed o he web e sion o
his a icle.)
Chap e 4
85
EGFP-exp essing neu al cell mo phology we e disce ned on p ima y co ical cul u es
ans ec ed wi h DP60L nioplexes a 14/1 mass a io (w/w) in Fig. 6-A1. Howe e , none
o hem we e NeuN+ (Fig. 6-A2). 72 h a e mice in ac anial injec ion o nioplexes,
immunochemis y o he c yop ese ed sec ions showed NeuN− (non-neu onal) cells
wi h glial mo phology, exp essing EGFP h oughou dend i ic p ocesses. In e es ingly,
cells in he wall o some essels we e EGFP+ as well.
4.4.Discussion
Gene he apy di ec ed o he CNS ep esen s a g ea challenge o b ing no mal gene
copies and co ec mu an gene de iciencies in many neu odegene a i e diseases
(Magui e e al., 2014). Howe e , in o de o apply his p omising echnology in o he
egula clinic p ac ice, sa e and e icien gene ca ie sys ems a e needed. Compa ed
wi h i al ec o , non- i al ec o s ha e se e al impo an ad an ages. Fo ins ance,
hey a e easie and cheape o p oduce, and he e is no p eexis ing immuni y o hese
ec o s. Addi ionally, hey a e no de i ed om pa hogens, and consequen ly show less
sa e y conce ns. Howe e , hei ans ec ion e iciency is lowe han hei i us-based
coun e pa s. The e o e, esea ch on his opic me i s special a en ion.
Based on he la e ing p ope ies showed ecen ly by niosomes o ans ec e inal cells
a e bo h in a i eal and sub e inal adminis a ion (Mashal e al., 2017; Pu as e al.,
2014; Ochoa e al., 2014), we elabo a ed ca ionic niosomes based on DOTMA,
polyso ba e 60 and lycopene o deli e DNA in o he b ain co ex o a s a e ce eb al
co ex adminis a ion. DOTMA is a highly wa e -soluble qua e na y ammonium sal
lipid ha has been ex ensi ely used o gene deli e y pu poses, due o i s abili y o
condense DNA (Zhang e al., 2012). Lycopene is a na u al ca o enoid ha when used
as “helpe ” lipid, enhances ans ec ion e iciency in e inal cells (Mashal e al., 2017).
Polyso ba e 60 is he majo componen o he niosome o mula ion, and i s
poly(e hylene glycol) s uc u e has been epo ed ha inc eases in i o ans ec ion
e iciency in CNS (Tang e al., 2003; Yao e al., 2012).
P io o pe o m any biological s udy, we cha ac e ized in Fig. 2 ou o mula ion in
e ms o size, supe icial cha ge, mo phology and capaci y o condense, elease and
p o ec DNA agains enzyma ic diges ion. Al hough he e is no a gene al ule abou he
Chap e 4
86
op imal pa icle size o o mula ions o each gene deli e y applica ion, i is gene ally
accep ed ha his pa ame e clea ly in luences he inal pe o mance, and ha in case
o niosomes, is a ec ed by he ca ionic lipid/DNA mass a io (Pu as e al., 2015). Size
o DP60L niosomes was a ound 100 nm. In e es ingly, when DNA was inco po a ed o
ob ain nioplexes a 6/1 a io, size inc eased up o 154 nm. Howe e , a highe a ios,
size dec eased, p obably due o he elec os a ic in e ac ions ha condense DNA mo e
e icien ly, and he e o e, dec eased PDI alues as well. Addi ionally, di e ences
obse ed in PDI alues could be due o di e en DNA opologies ound in nioplexes
(Che ng e al., 1999). Rega ding ZP alues, we ound a posi i e co ela ion wi h he
ca ionic lipid/DNA a io, which sugges s ha ca ionic DOTMA binds and neu alize he
nega i ely cha ged DNA (Paecha oenchai e al., 2012). Unde TEM obse a ion,
niosomes appea ed sphe ical, while nioplexes a 14/1 mass a io showed he e ogeneous
shapes, bu mos ly elonga ed. In e es ingly, a his a io, sphe ical niosomes we e also
disce ned (Fig. 2-B2), which could accumula e on he su ace o cell memb anes and
enhance DNA deli e y (Smi h e al., 1998; Song and Liu, 1998). To u he analyze
elec os a ic in e ac ions, we pe o med a gel e a da ion assay (Fig. 2-C), since an
op imum balance is equi ed o e icien gene deli e y (Paecha oenchai e al., 2012).
Despi e he incomple e DNA condensa ion obse ed, which could be a ec by di e en
he modynamic ac o s, kine ics mixing, o by he lycopene inco po a ion in o he
niosome o mula ion o he high aqueous solubili y o DOTMA (Maha o, 2005), all
ca ionic lipid/DNA a ios analyzed, we e able o p o ec plasmid DNA agains
enzyma ic diges ion.
Once physicochemical p ope ies o nioplexes we e analyzed, nex , we pe o med in
i o s udies o e alua e, ini ially, bo h ans ec ion e iciency and iabili y in NT2 cells.
These cells ep esen an in e es ing model o s udy he e iciency o gene deli e y
ec o s in o CNS due o hei capaci y o di e en ia e in o bo neu onal and glial cells
(Agi e e al., 2015). Unlike o he e a oca cinoma cell lines, he NT2 cells depic an
exclusi e commi men o a neu al lineage when exposed o e inoic acid (RA).
The e o e, i has been conside ed as a p omising human cell sou ce in s udies o cell in
i o he apeu ic applica ions in many neu odegene a i e diseases such as Pa kinson
disease (Caccio i e al., 2017). Acco dingly, we conside ed NT2 cells as an in e es ing
Chap e 4
87
model o s udy he e iciency o gene deli e y ec o s in o CNS. Addi ionally, hese
cells ep esen a p omising pla o m in cell-based gene deli e y as hey could be
gene ically-modi ied and hen ansplan ed (Tinsley and E iksson, 2004).
Da a ob ained in Fig. 3 e ealed ha ans ec ion e iciency inc eased in p opo ion o
he ca ionic lipid/DNA mass a io, eaching he peak a 14/1 a io (17% o cells we e
ans ec ed). Al hough he pe cen age o ans ec ed cells we e in e io o hose ob ained
wi h comme cially a ailable lipi ec amine®2000 (17% and 37%, espec i ely), iabili y
alues we e highe (90% and 83%, espec i ely). The epo ed low cy o oxici y o
nioplexes ep esen s an appealing ea u e o po en ial u he in i o applica ions, since
he in i o use o lipi ec amine®2000 is discou aged due o i s cy o oxici y (Yang e al.,
2014). To be e unde s and he ans ec ion p ocess media ed by DP60L nioplexes in
NT2 cells, we e alua ed he cellula up ake and he in acellula a icking o nioplexes,
since hose wo ac o s clea ly in luence on he inal pe o mance o gene deli e y
ca ie s (Pu as e al., 2015). Cellula up ake alues we e compa ed wi h
lipi ec amine®2000. As obse ed in Fig. 4, lipoplexes ob ained wi h comme cially
a ailable lipo ec amine®2000, we e mo e e icien ly in e nalized han DP60L nioplexes
(a ound 85% and 60%, espec i ely), which could explain he highe pe cen age o
EGFP exp ession in NT2 cells ans ec ed wi h lipo ec amine®2000. Di e ences
obse ed in cellula up ake be ween bo h o mula ions could be due o pa icula
opologies o complexes o speci ic in e ac ions o he complexes wi h cell memb ane
lipids (Che ng e al., 1999). In any case, pe cen age o ans ec ed cells wi h DP60L
nioplexes (17%, Fig. 3) we e clea ly in e io o he pe cen age o posi i e cells o
FITC-labeled DP60L nioplexes (60%, Fig. 4-B), which sugges he in luence o o he
biological e en s, such as in acellula a icking o endosomal scape, in he inal EGFP
exp ession (Ca da elli e al., 2016). The e o e, we analyzed h ee o he mos employed
cellula in e naliza ion pa hways such as cla h in-media ed endocy osis (CME),
ca eolae-media ed endocy osis (C ME) and mac opinocy osis (MPC). Al hough he e
is no a clea consensus ega ding he mos e icien endocy osis pa hway, he elease
o DNA in o he cy oplasm, and he e o e, he inal pe o mance, is clea ly a ec ed by
he cellula up ake p ocess (Nam e al., 2009). The esul s obse ed in Fig. 5 sugges ed
ha DP60L nioplexes we e in e nalized, mainly, by C ME and CME, while MPC had

Chap e 4
88
much less pa icipa ion in he cellula up ake p ocess. I is gene ally accep ed ha bo h
C ME and CME a e endocy osis ou es ha ans e gene ic ma e ial o la e
endosomes/lysosomes, whe e he acidic en i onmen deg ades he DNA, making
ans ec ion p ocess ine icien (Agi e e al., 2015). The e o e, he obse a ion o
nioplexes in he la e endosome (Fig. 5-A4) migh explain he ela i ely low ans ec ion
e iciency alues (17%) obse ed (Fig. 3), despi e he ac ha high numbe o NT2
cells (60%) cap u ed he complexes (Fig. 4). None heless, his hypo hesis needs u he
e i ica ion since he endosomal escape capaci y o nioplexes, i p esen , could e ade
deg ada ion. The e o e, in nex expe imen s, we e alua ed he abili y o nioplexes o
escape om deg ada ion in lysosomes. Se e al pa icle dependen endosomal escape
mechanisms ha e been epo ed in he li e a u e, being he p o on-sponge mechanism
one o he mos widely desc ibed (Va kouhi e al., 2011).
The e o e, we analyzed he pH-bu e ing capaci y o ca ionic DP60L niosomes.
As obse ed (Supplemen a y da a, Fig. 1), he inco po a ion o bo h ca ionic DOTMA
and lycopene lipids in o he niosome o mula ion, inc eased he pH-bu e ing capaci y
upon i a ion wi h 0.1 M HCl compa ed wi h niosomes elabo a ed only wi h
polyso ba e 60, which could sugges ha hose lipids could inc ease he p o on sponge
e ec , and he e o e, he endosomal scape capaci y o DP60L niosomes. Ano he
widely p oposed endosomal scape mechanism consis s on he des abiliza ion o he
endosomal memb ane by elec os a ic in e ac ions be ween he ca ionic nanopa icles
and he anionic lipids o he la e endosome memb ane, which could allow he DNA
elease o he cy oplasm (Va kouhi e al., 2011). To e alua e his endosomal scape
mechanism, we added DP60L nioplexes o anionic micelles made wi h phospha idyl
se ine (PS), ha simula ed he endosomal compa men , and he DNA elease om PS
micelles was e alua ed in an aga ose gel elec opho esis assay (Agi e e al., 2015). As
obse ed in Fig. 5, abou 40% o DNA was eleased o m DP60L nioplexes, in he
p esence o PS, micelles. Howe e , only 30% o DNA was eleased wi hou p e ious
incuba ion wi h he anionic PS micelles, which demons a es he capaci y o he DP60L
nioplexes o elease DNA once hey con ac he endosomal lipid bilaye memb ane. In
he case o lipoplexes based on lipo ec amine®2000, all DNA was condensed wi h he
o mula ion and up o 20% was eleased in he p esence o PS micelles.
Chap e 4
89
Nex , and p io o pe o m in i o s udies, we e alua ed ans ec ion e iciency o
DP60L nioplexes in p ima y co ical cul u es o a emb yos, since p ima y cells
no mally exp ess hei issue-speci ic ecep o s, and mimic in i o condi ions, whe e
di e en kind o neu ons and glial cells a e mixed o se up neu onal-glial ne wo ks. In
hese condi ions, we obse ed ha , appa en ly, only glial cells, exp essed EGFP (Fig.
6-A1). This assump ion was u he con i med by lack o NeuN+-immuno eac i i y in
EGFP exp essing cells (Fig. 6-A2). Such p e e en ial ans ec ion o glial cells could be
a ibu ed o hei highe mi o ic and/o phagocy ic ac i i ies (Scha e and S e ens,
2013).
In e es ingly, in in i o expe imen s pe o med by di ec in ac anial injec ion o
nioplexes, again, NeuN nega i e cells (neu oglia and cells in blood essel wall) we e
he only ones ans ec ed by DP60L nioplexes (Fig. 6-B). These esul s e eal he
incapaci y o nioplexes o ans ec neu on cells, p obably due o he impai ed up ake
and/o in acellula a icking (Be gen e al., 2008). The e o e, DP60L nioplexes could
be o g ea in e es o ans ec glial cells in he CNS in glia- ela ed neu ological
diso de s. Glial cells cons i u e o e 70% o he o al cell popula ion in he CNS, and
hey play a pi o al ole o he no mal de elopmen and unc ion o ne ous issue
(Al a ez e al., 2013; Fields and S e ens-G aham, 2002). Thei pe u ba ion is
associa ed wi h se e al neu ological diso de s such as; s oke, mul iple scle osis,
epilepsy, Alzheime ’s and Pa kinson’s diseases (Ba es, 2008; Milligan and Wa kins,
2009). The e o e, he p e e en ial ans ec ion o glial cells could be o g ea impo ance
in u u e applica ions in glia- ela ed neu ological diso de s. Addi ionally, cells in he
wall o some blood essels we e also ans ec ed wi h DP60L nioplexes (Fig. 6-B, whi e
a ow). T ans ec ion a his le el could be o g ea ele ance in ce eb o ascula
diseases, such as; s oke, ansien ischemic a acks, suba achnoid hemo hage o
ascula demen ia, jus o name a ew.
4.5.Conclusion
In summa y, we conclude ha non- i al ec o o mula ions based on niosome
nanopa icles, whe e DOTMA is he ca ionic lipid, lycopene he “helpe ” lipid and
polyso ba e 60 he non-ionic su ac an , p esen s sui able physicochemical p ope ies
Chap e 4
90
o gene deli e y applica ions in e ms o size, supe icial cha ge, polydispe si y, o
capaci y o p o ec gene ic ma e ial agains enzyma ic diges ion. In addi ion, such
o mula ion was able o ans ec e icien ly NT2 cul u ed cells, we e bo h cla h in and
ca eolae-media ed endocy osis pa hways p edomina ed o e mac opinocy osis,
exhibi ing endosomal scape p ope ies ha could explain he high p o ein exp ession
le els obse ed. P omising esul s ob ained in bo h p ima y co ical cul u es o a
emb yos and in in i o condi ions a e in ac anial injec ion open he doo o u u e
applica ion o such niosomes as e icien gene deli e y ools o he apeu ic ea men
o some degene a i e as well as malignan CNS diso de s.
4.6.Acknowledgemen s
This p ojec was suppo ed by he Basque Coun y Go e nmen (GIC15/85), Spanish
G an MAT 2015 -69976-C3-1, SAF2013-42347-R, and by Resea ch Chai “Bidons
Ega a”. The au ho s also wish o hank he in ellec ual and echnical assis ance om he
ICTS “NANBIOSIS”, mo e speci ically by he D ug Fo mula ion Uni (U10) o he
CIBER in Bioenginee ing, Bioma e ials, and Nanomedicine (CIBER-BBN) a he
Uni e si y o Basque Coun y (UPV/EHU). Technical and human suppo p o ided by
SGIke (UPV/EHU) is g a e ully acknowledged
4.7. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://
doi.o g/10.1016/j.ijpha m.2018.09.038.
Chap e 4
91
Supplemen a y igu e 1. pH bu e ing capaci y o assay o bo h DP60L and P60
niosomes.
4.8.Re e ences
Agi e, M., Ojeda, E., Za a e, J., Pu as, G., G ijal o, S., E i ja, R., Ga cia del Cano, G.,
Ba ondo, S., Gonzalez-Bu gue a, I., Lopez de Jesus, M., Salles, J., Ped az, J.L., 2015.
New insigh s in o gene deli e y o human neu onal p ecu so NT2 cells: a compa a i e
s udy be ween lipoplexes, nioplexes, and polyplexes. Mol. Pha m. 12, 4056–4066.
h ps://doi.o g/10.1021/acs.molpha maceu .5b00496.
Al a ez, J.I., Ka ayama, T., P a , A., 2013. Glial in luence on he blood b ain ba ie .
Glia 61, 1939–1958. h ps://doi.o g/10.1002/glia.22575.
A ia, N., Mashal, M., G ijal o, S., E i ja, R., Za a e, J., Pu as, G., Ped az, J.L., 2017.
S em cell-based gene deli e y media ed by ca ionic niosomes o bone egene a ion.
Nanomedicine S1549-9634(17)30200-9 [pii].
Ba es, B.A., 2008. The mys e y and magic o glia: a pe spec i e on hei oles in heal h
and disease. Neu on 60, 430–440. h ps://doi.o g/10.1016/j.neu on.2008.10.013.
Be gen, J.M., Pa k, I.K., Ho ne , P.J., Pun, S.H., 2008. Non i al app oaches o
neu onal deli e y o nucleic acids. Pha m. Res. 25, 983–998.
h ps://doi.o g/10.1007/s11095007-9439-5.
Caccio i, I., Ceci, C., Bianco, A., Pis i o, G., 2017. Neu o-di e en ia ed N e a2
cance s em cells encapsula ed in algina e beads: i s e idence o biological
Chap e 5
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Chap e 5
99
Gene deli e y o he a e ina by non- i al ec o s based on chlo oquine-
con aining ca ionic niosomes
Mohamed Mashala,1, Noha A iaa,b,c,1, Gema Ma ínez-Na a e ed,e, C is ina So o-Sánchezd,e,
Edua do Fe nándezd,e, San iago G ijal od, , Ramón E i jad, , Gus a o Pu asa,d, , Jose Luis
Ped aza,d,
a NanoBioCel G oup, Labo a o y o Pha maceu ics, School o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU), Paseo
de la Uni e sidad 7, 01006 Vi o ia-Gas eiz, Spain
b His ology and Cell Biology Depa men , Facul y o Medicine, Uni e si y o Alexand ia, Alexand ia, Egyp
c Depa men o Basic Sciences, The Ame ican Uni e si y o An igua-College o Medicine, Coolidge, An igua and Ba buda
d Ne wo king Resea ch Cen e o Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN), Vi o ia-Gas eiz, Spain
e Neu op o hesis and Neu oenginee ing Resea ch G oup, Miguel He nández Uni e si y, Elche, Spain
Ins i u e o Ad anced Chemis y o Ca alonia (IQAC-CSIC), Spain
Jou nal o Con olled Release 304 (2019) 181–190
ABSTRACT
The inco po a ion o chlo oquine wi hin nano o mula ions, a he han as a co-
ea men o he cells, could open a new a enue o in i o e inal gene deli e y. In his
manusc ip , we e alua ed he inco po a ion o chlo oquine diphospha e in o he ca ionic
niosome o mula ion composed o poloxame 188, polyso ba e 80 non-ionic
su ac an s, and 2,3-di ( e adecyloxy) p opan-1-amine (hyd ochlo ide sal ) ca ionic
lipid, o ans ec a e ina. Niosome o mula ions wi hou and wi h chlo oquine
diphospha e (DPP80, and DPP80-CQ, espec i ely) we e p epa ed by he e e se phase
e apo a ion echnique and cha ac e ized in e ms o size, PDI, ze a po en ial, and
mo phology. A e he inco po a ion o he pCMS-EGFP plasmid, he esul an
nioplexes -a diffe en ca ionic lipid/DNA mass a ios- we e u he e alua ed o
compac , libe a e, and secu e he DNA agains enzyma ic diges ion. In i o p ocedu es
we e achie ed in ARPE-19 cells o assess ans ec ion efficacy and in acellula
anspo a ion. Bo h nioplexes o mula ions ans ec ed efficien ly ARPE-19 cells,
al hough he cell iabili y was clea ly be e in he case o DPP80-CQ nioplexes. A e
sub e inal and in a i eal injec ions, DPP80 nioplexes we e no able o ans ec he a
e ina. Howe e , chlo oquine con aining ec o showed p o ein exp ession in many
e inal cells, depending on he adminis a ion ou e. These da a p o ide new insigh s
o e inal gene deli e y based on chlo oquine-con aining niosome non- i al ec o s.
Keywo ds: Niosomes, Non- i al ec o , Gene he apy, Re ina, Chlo oquine
Chap e 5
100
5.1.In oduc ion
Re inal degene a ion is a de as a ing ocula pa hology caused by unc ional
impai men o genes ela ed mainly o he pho o ansduc ion p ocess, he s uc u e and
me abolism o he e inal cells, and he ma u a ion p ocess o he mRNA needed o
syn he ize speci ic p o eins. One o he mos p omising al e na i es o ea e inal
diso de s like age- ela ed macula degene a ion [1], Lebe congeni al amau osis (LCA)
[2] e ini is pigmen osa [3] o cho oide emia consis s on he deli e y o a no mal copy
o he mu a ed genes o he affec ed cells by means o gene he apy echnology [4].
Since he success o i s RPE65-gene- eplacemen ials o LCA ype-2, u he
clinical ials o gene he apy ha e been conduc ed o o he de as a ing e inal
diso de s [5]. In mos o hose clinical ials, i al- ec o s ha e been used o deli e he
gene ic ma e ial. Among hem, adeno-associa ed i us (AAV) s and ou o hei sa e y
p o ile [6]. In ac , Lux u na, he i s gene he apy-based medicine app o ed by he
FDA in 2017 o he ea men o mu a ions in RPE65 gene linked o e ini is
pigmen osa and S a ga d disease, is based on such AAV. Howe e , he limi ed
ca ying capaci y o gene ic ma e ial, a ound 5 kb, hampe s hei ap-plica ion o deli e
genes ha o e pass such size o he e ina. Fo ins ance, ABCA4 and MYO7A genes,
whose mu a ions can be ela ed o S a ga d disease and Ushe Synd ome Type 1B,
espec i ely [7], ha e a size o a ound 7 kb. The e o e, he use o non- i al ec o s
ep esen s an in e es ing al e na i e, since he size o DNA ha can be inse ed in some
o hese o mula ions is heo e ically unlimi ed [8,9].
Despi e hei limi ed ans ec ion efficiency and ansien gene ex-p ession, non-
i al ec o s ha e eme ged as a p omising al e na i e o deli e gene ic ma e ial. Some
o he main ad an ages o such gene deli e y sys ems, in addi ion o hei highe
ca ying capaci y, include hei low cos o p oduc ion o hei capaci y o be easily
modi ied in o de o enhance hei pe o mance [10,11]. Hence, he esea ch ac- i i y
ela ed o he design and cha ac e iza ion o no el non- i al ec o o mula ions o
gene deli e y has conside ably inc eased [12]. Ca ionic niosomes a e sel -assembled
esicula nano ehicles, simila o liposomes, wi h encou aging p ope ies o gene
deli e y applica ions. To men ion a ew, he chemical s uc u e o niosomes makes i
possible o p o ide mo e s able and less cy o oxic o mula ions a a low cos [13]. The
Chap e 5
101
amphiphilic na u e o non-ionic su ac an s enable niosomes o ap bo h hyd ophobic
and hyd ophilic compounds [14].The ca ionic pa he e is he hyd ochlo ide sal o he
ca ionic lipid 2,3-di ( e a-decyloxy) p opan-1-amine (D). Such ca ionic lipid con ains
he ou pi o al componen s ha manage he gene ans ec ion p ocess: a pola head, a
backbone, a linke , and wo non-pola ails [15].
One o he key limi ing s eps in he ans ec ion p ocess is he endosomal
escape. Chlo oquine is a known endosomal dis up ing molecule and lysosomo opic
agen ha can c oss he blood e inal ba ie . Al hough chlo oquine has been used in
i o as a p e- ea men o cul u ed cells o acili a e gene deli e y [16], his s udy will
be he i s - o he bes o ou knowledge- o apply a chlo oquine-con aining nio-some
o mula ion in gene deli e y se ing. The inco po a ion o one o mo e o he ma e ials
a he molecula le el, wi hin he nano- o mula ion, can d ama ically affec he
ans ec ion p ocess unde in i o and in i o condi ions [12]. The ea e , scien is s
may ace a g ea challenge in he nea u u e o es a lib a y o diffe en ma e ials ha
can be inco po a ed wi hin he gene deli e y ehicles.
Based on he a o emen ioned (D) ca ionic lipid, wo niosome ehicles we e
o mula ed o e inal gene deli e y wi h wo non-ionic su ac an s [polyso ba e 80
(P80) and poloxame 188 (P)], in he absence/p esence o chlo oquine (CQ), e e ed
as DPP80 and DPP80-CQ, espec i ely (Fig. 1). The wo ehicles we e p epa ed by he
emulsi ica ion/sol en e apo a ion sys em and cha ac e ized in e ms o pa icle size,
polydispe si y index (PDI) and ze a po en ial. Then, he epo e pCMS-EGFP plasmid
was added a diffe en weigh a ios o ca ionic lipid o ob ain nioplexes. Such
DPP80/DPP80-CQ nioplexes we e u he cha ac e ized by size, PDI, cha ge,
mo phology, and he capabili y o compac , libe a e and p o ec he DNA om diges i e
enzymes. In i o compa a i e s udies o bo h ehicles in ARPE-19 cells we e achie ed
espec ing hei cellula up ake, ans ec ion efficiency, iabili y and in e naliza ion
mechanism. Finally, he wo o mula ions we e adminis e ed o a eyes ia in a i eal
and sub e inal injec ions, as a p obe o concep , o es ima e ans ec ion efficiency by
con ocal mic oscopy.
5.2.Ma e ials and me hods
Chap e 5
102
5.2.1. P oduc ion o ca ionic niosomes
The syn hesis o he hyd ochlo ide sal o m o he ca ionic lipid 2,3-di
( e adecyloxy) p opan-1-amine (D) was pe o med as desc ibed in he li e a u e, wi h
sligh modi ica ions o he labo a o y p o ocol [17]. Niosomes we e composed by
modi ied e e se-phase e apo a ion app oach [18]. Concisely, 5 mg (0.1% w/ ) o he
lipid was dispe sed in 1 ml o dichlo ome hane (o ganic phase). Subsequen ly, 5 ml
milliQ wa e con aining 12.5 mg (0.25% w/ ) poloxame 188 (P) (Sigma-Ald ich,
Mad id, Spain), 12.5 mg (0.25% w/ ) polyso ba e 80 (P80) (Sigma-Ald ich, Mad id,
Spain) and 2.5 mg (0.05% w/ ) chlo oquine diphospha e (CQ) (Sigma-Ald ich,
Mad id, Spain) we e added o he o ganic phase.
Fig. 1. Chemical s uc u e o he componen s o DPP80 and DPP80-CQ niosomes.
(A) Polyso ba e80, (B) Chlo oquine diphospha e sal , (C) Poloxame 188 and (D)
ca ionic lipid (DTPA-Cl).
The emulsions we e ob ained by sonica ion o he mix u e o 50 s a 45 W
(B anson Soni ie 250®, Danbu y, USA). Di-chlo ome hane was elimina ed om
emulsions by dissipa ion unde magne ic s i ing o 2 h, ende ing he ca ionic
niosomes in he aqueous medium. The mola a ios o bo h DPP80 and DPP80-CQ
o mula ions we e, 1.9/0.3/1.9 and 1.9/0.3/1.9/1, espec i ely.
Chap e 5
103
5.2.2. Plasmid p opaga ion and elabo a ion o nioplexes
The p o ocols o p opaga ion, pu i ica ion, and quan i ica ion o pCMS-EGFP
plasmid (5541 bp, Plasmid Fac o y, Biele eld, Ge many), ha e been desc ibed
p e iously [12]. The nioplexes (niosome/DNA complexes) o bo h DPP80 and DPP80-
CQ we e o med by mixing an adequa e olume o pCMS-EGFP plasmid s ock solu ion
(0.5 mg/ml) wi h a ious amoun s o he niosome suspension (1 mg ca ionic lipid/ ml)
o ge diffe en ca ionic lipid/DNA mass a ios (w/w). To enhance he elec os a ic
in e ac ion, he nioplexes mix u e was allowed o se le o 30 min a oom empe a u e.
5.2.3. Cha ac e iza ion o niosomes/nioplexes
Dynamic ligh sca e ing (DLS) echnique was used o es ima e bo h pa icle
size and polydispe si y index (PDI) measu emen s (Mal e n Ze asize Nano ZS, UK).
Pa icle size, was de e mined by cumula i e analysis o he eco ded hyd odynamic
diame e . Lase Dopple Velocime y (LDV) was used o assess ze a po en ial (ZP).
Samples we e dispe sed in a 0.1 mM NaCl solu ion. T iple measu emen s we e ca ied
ou o all samples. The mo phology o bo h niosomes and nioplexes was es ima ed by
ansmission elec on mic oscopy (TEM). Sho ly, on o glow-discha ged ca bon coa ed
g ids, 5 μl sample was allowed o adhe e on he su ace o 60 s. Samples we e
examined unde TEM, Tecnai G2 20 Twin (FEI, Eindho en, The Ne he lands). In a
b igh - ield image mode, he ope a ion was done wi h an accele a ing ol age o 200
keV. Digi al images we e cap u ed by an Olympus SIS Mo ada di-gi al came a.
Niosomes' abili y o compac , libe a e and sa egua d DNA om enzyma ic diges ion
was assessed by aga ose gel elec opho esis s udies. Nioplexes samples (200 mg o
plasmid DNA/20 μl) we e com-pa ed o naked (uncomplexed) DNA. The aga ose gel
(0.8% w/ ) was imme sed in a T is–ace a e–EDTA buffe , and he DNA samples we e
un on he gel o 30 min a 120 V. Nex , aga ose gel was s ained wi h GelG een®. A
digi al ChemiDoc™ MP Imaging Sys em (Bio-Rad, Mad id, Spain) was used o band
obse a ion. 20 μl o a 2% SDS solu ion (Sigma-Ald ich, Mad id, Spain) was added o
he samples o es ima e he libe a ion o DNA om bo h DPP80 and DPP80-CQ
ehicles a diffe en ca ionic lipid/DN mass a ios. In addi ion, DNase I (Sigma-Ald ich,
Mad id, Spain) was added a a concen a ion o 1 uni o DNase I/2.5 μg DNA o

Chap e 5
104
e alua e he p o ec i e abili y o bo h ehicles agains enzyma ic diges ion. Then, he
samples we e incuba ed a 37 °C o 30 min and a 2% SDS solu ion was added o
e alua e i eleased he DNA om he ehicles wad p o ec ed om he enzyma ic
diges ion.
5.2.4. In i o ans ec ion expe imen s
ARPE-19 cells, pu chased om he Ame ican Type Cul u e Collec ion (ATCC,
CRL 2302®), we e seeded in 24-well pla es a a densi y o 8 × 104 cells/well, wi h 500
μl o comple e medium, o med o D-MEM/F-12 con aining 10% e al bo ine se um
(Gibco®, Cali o nia, USA). Then, a a con luence le el o 70–80%, he media was
emo ed, and cells we e exposed o nioplexes a diffe en ca ionic lipid/DNA mass
a ios (w/w) (1.25 μg DNA/well) dispe sed in se um ee Op i-MEM® solu ion
(Gibco®, Cali o nia, USA) a 37 °C o 4 h. Subsequen ly, ans ec ion medium was
emo ed, and cells we e ho oughly washed 3 imes wi h PBS. Then, cells we e
cul u ed in 1 ml o comple e medium and allowed o g ow o u he 72 h un il
luo escence mic oscopy imaging (Nikon TSM) and low cy ome y analysis
(FACSCalibu ™, BD Biosciences, USA) we e done. FL1 (530/30) was used o de ec
EGFP-exp essing ans ec ed cells, and FL3 (670) was used o de ec P opidium Iodide-
s ained dead/dying cells. Un ans ec ed cells we e used as nega i e con ol o all
expe imen s, while cells ans ec ed wi h Lipo ec amine™ 2000 (In i ogen,
Cali o nia, USA), acco ding o manu ac u e 's p o ocol, we e conside ed as posi i e
con ols. 10.000-ga ed e en s we e acqui ed and analyzed using Flowing So wa e
2.5.1. Da a ep esen he mean (± SD) o h ee independen expe imen s, each o hem
pe o med in iplica e.
5.2.5. Cellula up ake and endocy osis mechanism s udies
FITC-labeled (pCMS-EGFP) plasmid (Da eBio, Mad id, Spain) was used
ins ead o pCMS-EGFP plasmid o es ima e he cellula up ake o he ehicles. The
same p o ocol desc ibed in he p e ious 5.2.4 sec ion, was used o incuba e and
main ain ARPE-19 cells, and o e alua e cellula up ake. A e emo al o he
ans ec ion medium and mul iple washes o he pla es wi h PBS, he cells we e assayed
Chap e 5
105
by FACSCalibu low cy ome e . The nega i e con ol cells we e ans ec ed wi h
naked DNA, and he pe cen age o FITC-posi i e cells ep esen ed he cellula up ake
alues. Each specimen was assayed in iplica e. Diffe en up- ake inhibi o s we e used
o es ima e he endocy osis mechanism o ehicles. Genis ein, chlo p omazine
hyd ochlo ide, me hyl-β-cyclo-dex in and wo mannin we e used as inhibi o s o
ca eolae-media ed endocy osis (C ME), cla h in-media ed endocy osis (CME), bo h
(C ME and CME) and mac opinocy osis (MPC), espec i ely. Nioplexes a 10/1
ca ionic lipid/DNA mass a io we e complexed wi h pCMS-EGFP plasmid, and
ollowed he same p o ocol desc ibed in he p e ious 5.2.4 sec ion o ans ec ARPE-
19 cells. P io o he addi ion o nioplexes, cells we e incuba ed wi h ei he 200 μM
genis ein o 30 min, o wi h 5 mM me hyl-β-cyclodex in, 50 nM wo mannin, o wi h
5 μg/ml chlo p omazine hyd ochlo ide o 60 min. Cells we e incuba ed wi h se um-
ee Op i-MEM® solu ion o 4 h a 37 °C. Subsequen ly, cells we e ca e ully washed
wi h PBS a e emo al o he ans ec ion medium. Then, comple e medium was
added, and cells we e incuba ed o g ow o a u he 72 h un il low cy ome e analysis
was done o de e mine he ans ec ion efficiency. Each specimen was analyzed in
iplica e.
5.2.6. Buffe ing capaci y o niosomes
The buffe ing capaci y o bo h DPP80 and DPP80-CQ niosomes was assayed
by olume ic analysis. B ie ly, 10 ml o mula ion samples we e i a ed wi h aliquo s
o 100 μl 0.1 M HCl solu ion, and he changes in pH alue we e moni o ed by a pH
me e (CRISON, GLP 21, Ba celona, Spain).
5.2.7. In i o s udies
In a i eal (4 μl con aining 100 ng o pDNA) and sub e inal injec ion (1 μl
con aining 25 ng o pDNA) o bo h DPP80 and DPP80-CQ nioplexes suspension we e
pe o med in o ou adul emale Sp ague–Dawley a s (6–7 weeks old, 200–300 g
weigh ) pe o mula ion. Expe imen s we e done acco ding o he Spanish and
Eu opean Union egula ions o he use o animals in esea ch and he Associa ion o
Resea ch in Vision and Oph halmology (ARVO) s a emen , as de-sc ibed in he
Chap e 5
106
li e a u e [12]. To deli e nioplexes o he sub e inal space, a ben 33-gauge needle was
in oduced h ough a scle o omy (1–2 mm) pos e io o o a se a a and in a angen ial
di ec ion owa d he pos e io e inal pole along he sub e inal space. Success ul ad-
minis a ion was con i med by he appea ance o a pa ial e inal de achmen by di ec
oph halmoscopy o he eye undus h ough he ope a ing mic oscope (Zeiss OPMI®
pico; Ca l Zeiss Medi ec GmbH, Jena, Ge many). The un ea ed igh eyes we e
injec ed only wi h he ehicles and se ed as nega i e con ols.
Ra s we e sac i iced and pe used wi h 4% pa a o maldehyde (PFA) a e 72 h and
eyes we e emo ed, opened a he co nea and imme sed in PFA. Fo whole moun s,
e ina was dissec ed om he eyecup and la ened on o Supe os glass slides
(Supe os Plus, Fishe Scien i ic). Fo c yosec ions, he eyes we e c yop o ec ed in
suc ose and embedded in Tissue-Tek® OCT (op imum cu ing empe a u e). The eyes
we e c yosec ioned a 16 μm and ans e ed di ec ly on o mic oscope slides
(Supe os , Fishe Scien i ic).
Fo immunohis ochemis y, whole moun s o e inal sec ions we e washed and
blocked wi h 10% bo ine se um albumin and 0,05% i on in PBS o 1 h (c yosec ions)
o 2 h (whole moun s). Bo h sec ions and whole moun s we e incuba ed o e nigh a 4
°C wi h p ima y an ibodies: abbi an i-NeuN (Me ck Millipo e, MA, USA), abbi
an i- eco e in (Me ck Millipo e, MA, USA), abbi an i-P o ein kinase C (PKC, San a
C uz Bio echnology) and abbi an i-GFAP (San a C uz Bio echnology). Samples we e
insed and incuba ed wi h Alexa Fluo 555 donkey an i abbi (The mo ishe Scien i ic)
and coun e s ained wi h Hoechs 33342 (The mo ishe Scien i ic). Finally, whole
moun s and sec ions we e moun ed wi h an i ade moun ing meédium and e alua ed
wi h a Leica TCS SPE spec al con ocal mic oscope (Leica Mic osys ems GmbH,
We zla , Ge many).
5.2.8. S a is ical analysis
INSTAT p og am (G aphPad So wa e, San Diego, CA, USA) was used o
pe o m he s a is ical analysis. Diffe ences be ween g oups a signi icance le els o
Chap e 5
107
95% we e calcula ed by he ANOVA and he S uden 's - es . In all cases, P alues <
.05 we e ega ded as signi ican . No mal dis ibu ion o samples was assessed by he
Kolmogo o -Smi no es and he homogenei y o he a iance by he Le ene es .
Nume ical da a we e p esen ed as mean ± SD.
Table 1
Physical ea u es o bo h DPP80 and DPP80-CQ niosomes in e ms o size (nm),
Polydispe si y index (PDI), and ze a po en ial (mV). The alues exempli y he mean ±
SD (n = 3).
Size (nm)
Ze a po en ial (mV)
PDI
DPP80
90.41 ± 0.65
44.3 ± 1.48
0.42 ± 0.01
DPP80-CQ
118.18 ± 1.46
28.9 ± 7.73
0.13 ± 0.02
5.3.Resul s
5.3.1. Cha ac e iza ion o niosomes/nioplexes
Bo h niosome ehicles we e p epa ed by mixing [polyso ba e 80 (P80) and
poloxame 188 (P)] non-ionic su ac an s and ca ionic lipid (D). In he
absence/p esence o chlo oquine (CQ), niosomes we e e e ed as DPP80, o as DPP80-
CQ, espec i ely (Fig. 1). Bo h niosomes we e p epa ed by he emulsi ica ion/sol en
e apo a ion me hod and we e cha ac e ized in e ms o pa icle size, ze a po en ial (ZP)
and polydispe si y index (PDI) as shown in Table 1. The inco po a ion o chlo oquine
in o he DPP80 niosome o mula ion inc eased he size o hose niosomes om 90
o118 nm. Mo eo e , upon chlo oquine addi ion, he ZP alues dec eased ema kably
o 29 mV in DDP80-CQ niosomes compa ed o 44 mV in DPP80 niosomes.
In e es ingly, he addi ion o chlo oquine also dec eased he PDI alue om 0.42 in
DPP80 o mula ion o 0.13 in DPP80-CQ o mula ion.
Fig. 2 illus a es he physicochemical cha ac e iza ion o DPP80 and DPP80-CQ
nioplexes. In Sec ion 2-A, he size and ZP alues o bo h nioplexes a diffe en a ios
Chap e 5
114
o polyca ion-DNA complexes enhanced he exp ession le el o he deli e ed genes in
bo h in i o and in i o condi ions a doses below he known oxici y le els [22]. P80
has been epo ed o ac as a co-emulsi ie along wi h P, in d ug and gene deli e y
endea o s [23]. Mo eo e , he encou aging p ope ies o P80 c ea e a s e ic ba ie ha
e ades he agg ega ion o nano- esicles, enhances he cell ole ance [11], and imp o es
ans ec ion efficiency due o he p esence o polye hylene glycol (PEG) chains in i s
s uc u e [24]. Howe e , he abili y P o o m ne wo k s uc u es migh be mo e sui able
han P80, i used wi h wa e -soluble ca ionic lipids, o enhance hei lexibili y and
du abili y [25]. In such case, a mix u e o wo speci ic ypes o non- ensioac i e
molecules could p o ide a syne gis ic enhancemen o nano- esicle s abiliza ion [26].
Rega ding he ca ionic lipid, he high solubili y o he D-Cl sal enhances
biodis ibu ion o lipid/plasmid complexes, and he e o e, ans ec ion efficiency [27].
Howe e , in a p e ious s udy, we obse ed ha he solubili y o ca ionic lipid can
d ama ically shi he ans ec ion esul s acco ding o he ype o he cells and he way
o o mula ion. In ha s udy, he DTPA ca ionic lipid (non-sal o m) succeeded o
ans ec e inal cells in i o condi ions [11], while in such men ioned s udy, he sal
o m ailed o ans ec e inal cells in i o. In e es ingly, he same o mula ion wi h
he same sal o m o ca ionic lipid (DPP80) succeeded o ans ec ce eb al co ical
cells in i o [28]. S ikingly, bo h sal /non-sal o ms o he ca ionic lipid we e able o
ans ec ARPE-19 cells in i o condi ions. In any case, he non-sal o m was supe io
in e ms o ans ec ion and cell iabili y. This con adic ion emphasizes he lack o
co ela ion be ween he in i o and in i o ans ec ion condi ions and he impo ance
o he o mula ion a physical le el.
To emphasize he impac o chlo oquine, DPP80 and DPP80-CQ niosomes we e
elabo a ed and compa ed. The cha ac e iza ion da a o bo h niosomes we e analyzed
(Table 1). The inco po a ion o chlo oquine sligh ly inc eased he size o niosomes by
abou 28 nm, and educed bo h PDI (abou 69% dec ease) and ZP (abou 34% dec ease).
D ug/gene deli e y ehicles a e gene ally a o ed by small poly-dispe si y alues [29].
The posi i e ZP alues (> +25 mV) de ec ed o bo h niosomes would e lec a

Chap e 5
115
po en ially long-las ing s abili y. Once he niosomes we e cha ac e ized in e ms o
size, PDI, and ze a po en ial, nioplexes we e elabo a ed wi h he pCMS-EGFP plasmid
a a ious ca ionic lipid/DNA mass a ios by adding he epo e plasmid o he
niosomes and no he opposi e o ensu e p ope condensa ion p ocess [30].
Fig. 5. Re inal c oss sec ions mic og aphs ob ained by con ocal mic oscopy 3 days pos
sub e inal injec ion o DPP80-CQ nioplexes (A–D). EGFP p o ein was obse ed
mainly in GCL (whi e a ows), pho o ecep o s (yellow a ows) and RPE cells (blue
a ows). Re inal sec ions we e s ained wi h an ibodies agains NeuN(A), eco e in (B,
D) and p o ein kinase C (C). The cell nuclei we e coun e s ained wi h Hoechs 33342.
GCL, ganglion cell laye ; INL, inne nuclea laye ; ONL, ou e nuclea laye ; OS,
pho o ecep o ou e segmen Scale ba s: 20 μm. (Fo in e p e a ion o he e e ences o
colou in his igu e legend, he eade is e e ed o he web e sion o his a icle.)
The ZP o DPP80 nioplexes was clea ly lowe when compa ed o he same
niosomes wi hou chlo oquine (Fig. 2-A). On he o he hand, ZP o chlo oquine-
con aining nioplexes (DPP80-CQ) oscilla ed wi hin a na owe ange (19–25 mV) in
compa ison o DPP80-CQ niosomes (29 mV). Gene ally, he compac ion o DNA is
imp o ed when 90% o he cha ge is compensa ed in an aqueous solu ion [31].
S ikingly, a 8/1 and 10/1 mass a ios o DPP80 and DPP80-CQ, espec i ely, ZP
alues luc ua ed wi hin a na ow ange (23–27 mV) which ep esen s a small educ ion
Chap e 5
116
in ZP o DPP80-CQ compa ed o ZP o niosomes (29 mV). This sugges s a
spon aneous elec os a ic in e -ac ion o pDNA wi h DPP80-CQ niosomes a 10/1 mass
a io which could be explained by a di ec in e ac ion o chlo oquine wi h pDNA.
Rega ding PDI alues o nioplexes, an ob ious effec o chlo oquine addi ion a all
a ios s udied abo e 4/1 was obse ed, as PDI alues dec eased in compa ison o
DPP80 o mula ion (Supplemen a y Table 1). The elec on mic og aphs illus a ed a
disc e e, almos sphe ical mo phology and absence o agg ega es in DPP80 complexes
(Fig. 2-B1). By con as , DPP80-CQ nioplexes appea ed as clus e s o mul ilamella
plana s uc u es ha o m s ing-like colloidal agg ega es (Fig. 2-B2). The lamella
spacing was a ound 5.5–6 nm, sugges ing ha he pDNA s ands we e complexed wi h
he ca ionic lipid bilaye s [15]. Simila ly, many mix u es o neu al lipids (as DOPC
and DOPE), along wi h ca ionic lipids (as DOTAP), ex ensi ely used o gene deli e y
pu poses, a e known o o m lamella complexes wi h DNA [32].
The aga ose gel e a da ion assay showed ha bo h niosomes, a all s udied ca ionic
lipid/DNA a ios, we e able o condense, elease and p o ec he DNA om enzyma ic
diges ion (Fig. 2-C). O no e, he ela i ely lowe DNA condensa ion, obse ed by he
chlo oquine-con aining o mula ion (Fig. 2-C2), did no hampe he elease o he
p o ec ion o he condensed DNA, which is o u mos impo ance du ing he
ans ec ion p ocess. Any change in condensa ion efficiency migh affec he pa e n
and opology o spa ial DNA con igu a ion. E en mo e, he s a e o DNA condensa ion
can be affec ed by bo h he ype and he con en o he su ac an o o he addi i es as
chlo oquine. The e o e, he ine- uning o such molecules could be o impo ance o
un eil he mechanism o condensa ion o diffe en ypes o DNA molecules wi hin
diffe en nano- esicles. E en a high concen a ions o chlo oquine, 100 μg/ml, ARPE-
19 cells appea ed heal hy wi h good iabili y, despi e he appea ance o many acuoles
in he cy oplasm (Supplemen a y Fig. 1). The ans ec ion efficiency in i o, ARPE-19
cells, luc ua ed wi hin a small ange in bo h ec o s a all mass a ios s udied (Fig. 3).
Howe e , he cell iabili y was in a o o DPP80-CQ (Fig. 3-A). No ewo hy,
chlo oquine inhibi s lysosomal enzymes by inc easing he pH o he lysosomes and
dis u bing hei usion wi h au ophagosomes, hus inhibi s au ophagy [33]. Mo eo e ,
Chap e 5
117
chlo oquine and i s au ophagy inhibi ing de i a i e, hyd oxychlo oquine, a e bo h
FDA-app o ed agen s [34]. Acco ding o he cell ype o he s a e o s ess, au ophagy
migh p o ec o p omo e cell dea h in he eye [35].
Fig. 6. Con ocal luo escence mic og aphs o whole moun (A, B) and c oss-sec ions
(C, D) o he e ina 3 days a e in a i eal adminis a ion o DPP80-CQ nioplexes.
EGFP exp ession can be obse ed in bo h GCL (A, C and D, whi e a ows) and INL (B
and C, yellow a ows). In e es ingly, some p o ein exp ession was also obse ed in
OPL (C, blue a ows). Whole moun and e inal sec ions we e s ained wi h NeuN (A-
D). The cell nuclei we e coun e s ained wi h Hoechs 33342 (blue). GCL, Ganglion cell
laye ; INL, inne nuclea laye ; ONL, ou e nuclea laye ; OPL, ou e plexi o m laye .
Scale ba s: 20 μm. (Fo in e p e a ion o he e e ences o colou in his igu e legend,
he eade is e e ed o he web e sion o his a icle.)
This mu able na u e o au ophagy migh be he eason o he inc eased cell iabili y
obse ed wi h DDP80-CQ o mula ion in compa ison wi h i s chlo oquine- ee
Chap e 5
118
coun e pa , DPP80. Gene ally, cell iabili y and me abolism o ARPE-19 cells a e
ela i ely unaffec ed by he concen a ions o chlo oquine be ween 10 and 30 μg/ml,
hough affec ed in a dosage-dependen ashion a e wa d [36]. To analyze whe he he
enhanced cell in e naliza ion o nioplexes was among he effec s ha chlo oquine could
ha e on niosome o mula ions, we de e mined he pe cen age o ARPE-19 cell up ake
o bo h DPP80 and DPP80-CQ o mula ions a he mass a ios o bes ans ec ion
efficiency, 8/1 o DPP80 and 10/1 o DPP80-CQ (Fig. 4).
In e es ingly, low cy ome y s udies showed ha chlo oquine inco po a ion
had an insigni ican effec on he pe cen age o cellula up ake when compa ed o he
DPP80 o mula ion (Fig. 4-A). Such obse a ion is mos p obably due o he indiffe en
su ace cha ge o bo h nioplexes a he a o emen ioned mass a ios (22.5 ± 7.3 and 25.3
± 2.5 o DPP80 and DPP80-CQ, espec i ely, P > .05). The simila up ake pe cen ages
in such a ios could jus i y he unal e ed ans ec ion esul s depic ed p e- iously (Fig.
3-A). The ans ec ion efficiency can be ma kedly affec ed by he mechanism o
endocy osis. Consequen ly, we s udied h ee o he mos ac i e cellula in e naliza ion
pa hways: cla h in-media ed endocy osis (CME), ca eolae-media ed endocy osis
(C ME) and mac o-pinocy osis (MPC). The esul s obse ed in Fig. 4-B sugges ed ha
DPP80-CQ nioplexes we e in e nalized mainly by MPC, while C ME and CME had
less pa icipa ion in he cellula up ake p ocess. Due o i s abili y o in e nalize la ge
s uc u es, mac opinocy osis pa hway has been p oposed o media e he in e naliza ion
o non- i al gene deli e y ehicles [37]. Mo eo e , MPC is conside ed as he majo
pa hway esponsible o DNA ans ec ion in ce ain cell ypes [38]. In con a y, DPP80
nioplexes we e in e nalized mainly by CME and, o a lesse ex en , by MPC, while
C ME had a much less pa icipa ion in he cellula up ake p ocess. Howe e , he mino
luc ua ion in ans ec ion efficiency be ween he wo nioplexes could be due o limi ed
a ia ions be ween he wo main diffe en mechanisms o in e naliza ion (CME o
DPP80 and MPC o DPP80-CQ). The deli e y o gene ic ma e ial by C ME and CME
passes h ough la e endosomes/lysosomes, which in-c eases he haza ds o DNA
deg ada ion and lowe s he ans ec ion efficiency [39]. So, an expec ed i ial effec o
C ME and especially CME pa hways could explain he high pe cen ages o EGFP
exp ession in ARPE cells by bo h nioplexes (Fig. 3), compa ed o lipo ec amine® 2000
Chap e 5
119
(app oxima ely, 80% and 75% o lipo ec amine®2000 o DPP80 and DPP80-CQ,
espec i ely).
A e wa ds, we analyzed he pH-buffe ing capaci y o bo h niosomes (Fig. 4-
C). The inco po a ion o chlo oquine in o he niosome o mula ion inc eased he pH-
buffe ing capaci y upon i a ion wi h 0.1 M HCl, compa ed o he niosomes elabo a ed
wi hou chlo oquine (a pH alues > 2). Though, he e was no change in he buffe ing
capaci y when he pH was < 2 o bo h niosomes. Chlo oquine migh induce endosomal
and lysosomal escape ia he p o on sponge effec [40]. This esul could sugges ha
chlo oquine-con aining o mula ion could inc ease he p o on sponge effec , and
he e o e, he endosomal escape capaci y o DPP80-CQ niosomes. Howe e , as he
p edominan mechanism o in e naliza ion o DPP80-CQ was nei he C ME no CME,
he impac o he p o on sponge effec o chlo oquine on he ans ec ion efficiency was
insigni ican .
Based on he p e iously men ioned physicochemical and in i o biological
esul s, we we e en husias ic o pe o m a p elimina y in i o s udy o e alua e he
ans ec ion efficiency o ou o mula ions, DPP80-CQ in pa icula , in a e inae a e
bo h sub e inal (Fig. 5), and in a i eal injec ions (Fig. 6). Sub e inal injec ion is a
well-known clinical ou e o deli e gene ic/d ug ma e ial o he back o he eye. In
addi ion, i enables di ec con ac o he injec ed nucleic acids wi h he ou e e inal
laye s, pho o ecep o s and RPE cells. No ewo hy, clinical ials o ea many inhe i ed
e inal diseases such as LCA ype 2 used he sub e inal injec ion ou e [41]. Howe e ,
i is less desi able han he in a i eal ou e due o he possible complica ions; such as
e inal de achmen o he localized side effec s a ound he si e o injec ion. Gene ally,
IV injec ion is mo e widely applicable in he clinical p ac ice due o i s abili y o deli e
gene ic ma e ial o a la ge e inal su ace, in addi ion o less su gical auma compa ed
o he SR ou e [42].
Su p isingly, DPP80 did no induce any ans ec ion o e inal cells in i o a e
bo h sub e inal o in a i eal injec ions (Supplemen a y Fig. 2), whe eas he

Chap e 5
120
chlo oquine-con aining o mula ion, DPP80-CQ did (Figs. 5 and 6). The lack o
co ela ion be ween in i o and in i o ans ec ion esul s has been widely epo ed
as i is a con ex -dependen ma e [12].
Based on p e ious physicochemical and in i o biological esul s, we we e
en husias ic o pe o m a p elimina y in i o s udy o e alua e he ans ec ion
efficiency o ou o mula ions, DPP80-CQ in pa icula , in a e inae a e sub e inal
(Fig. 5) and in a i eal injec ions (Fig. 6). Sub e inal injec ion is a well-known clinical
iable ou e o deli e gene ic ma e ial o he eye. I enables di ec con ac o he
injec ed nucleic acids wi h he ou e e inal laye s, pho o ecep o s and RPE cells.
No ewo hy, clinical ials o ea many inhe i ed e inal diseases such as LCA ype 2
use sub e inal injec ion [41]. Howe e , i is less desi able han he IV ou e due o he
possible complica ions such as e inal de achmen o he localized effec a ound he si e
o injec ion. Gene ally, in a i eal injec ion is mo e widely applicable in he clinical
p ac ice due o i s abili y o deli e gene ic ma e ials o a la ge e inal su ace and
ad an ages o less su gical auma compa ed o he SR ou e [42].
Sub e inal adminis a ion allows di ec con ac o gene ic ma e ial wi h RPE
cells and ou e laye o he e ina. Al hough his ou e o adminis a ion is highly
effec i e o locally ans ec cells close o he si e o he injec ion, he occasionally
obse ed side effec s, ela ed o his in asi e ou e, such as e inal de achmen ,
hemo hages o al e a ions in RPE cells can hampe i s p ac ice [43]. In any case, sub-
e inal injec ions ha e been widely used on clinical ials o ea some de as a ing
gene ic diso de s o he e ina epo ing excellen ou comes [44]. In addi ion, he
ecen ly FDA/EMA-app o ed Lux u na medicine
o deli e heal hy copies o he RPE65 gene o he e ina is adminis e ed by sub e inal
injec ion. In ou in i o expe imen s, a e sub e inal adminis a ion o nioplexes, we
obse ed localized EGFP exp ession, mainly in some pho o ecep o and RPE cells,
close o he injec ion si e. T ans ec ion a his le el can ha e clinical ele ance because
mu a ions o > 200 genes in RPE cells/pho o ecep o s a e ela ed o ele an ge-ne ic
Chap e 5
121
diso de s o he e ina such as; Lebe congeni al amau osis, e ini is pigmen osa, and
S a ga d disease, o name jus a ew ones [45].
Compa ed o sub e inal injec ion, in a i eal injec ion ep esen s an in e es ing
al e na i e o deli e gene ic ma e ial o he back o he eye, and he e o e o access
e inal s uc u e. I is a less in asi e ou e, mo e easily o pe o m, and highe doses
can be deli e ed [46]. Consequen ly, la ge e inal su aces can be ans ec ed by his
ou e o adminis a ion [47]. When we adminis e ed 4 μl o DPP80-CQ nioplexes by
in a i eal injec ion, he inne laye s o he e ina (GCL and INL) we e mainly
ans ec ed as obse ed in Fig. 6. (whi e and yellow a ows, espec i ely). T ans ec ion
a his le el can be o clinical ele ance in ea men o de as a ing ocula pa hologies
ha comp omise he unc ion o ganglion cells as glaucoma [48]. In e es ingly, EGFP
exp ession was also disce ned in he OPL (Fig. 6-C, blue a ows) which sugges s ha
nioplexes pa ially diffused, no only h ough he i eous whe e hey we e
adminis e ed, bu also h ough he inne e inal laye s un il each he OPL. T ans ec ion
o he ou e laye s o he e ina by in a i eal adminis a ion o non- i al ec o s
ep esen s a g ea challenge o he scien i ic communi y, since can a oid he sub e inal
injec ions and he co esponding side effec s commonly associa ed o such injec ion.
Un o una ely, chlo oquine, like o he endoly ic agen s, has been ound o be
cy o oxic in se e al p e-clinical o clinical ials [49]. Chlo oquine passes he blood-
e inal ba ie and is oxic o he e ina. Ne e heless, such e inal oxici y is ela ed o
la ge doses and long- e m use o chlo oquine [50]. In his s udy, a 10/1 ca ionic lipid
/DNA mass a io, he inal concen a ion o chlo oquine was only 25 μg/ml which did
no induce any signi ican cy o oxici y in acco dance wi h Chen e al, [36]. The affini y
o e inal cells o he modi ied sal o m o he ca ionic lipid, in addi ion o he a o able
p ope ies o P and P80, along wi h he effec o chlo oquine, aise he possibili y o
a ge di e en e inal cell laye s sa ely and effec i ely a e bo h sub e inal and
in a i eal adminis a ions.
Chap e 5
122
5.5.Conclusions
The addi ion o chlo oquine o a niosome o mula ion e ained i s unc ionali y
in i o, bu mos impo an ly, enhanced i s ans ec ion abili y in i o. This wo k
highligh s he use o chlo oquine as a buil -in componen in he gene deli e y ehicles
o e ade i s oxici y and o p o ide new insigh s in o he u u e o e inal gene he apy.
5.6.Acknowledgemen s and disclosu es
This p ojec was suppo ed by he Basque Coun y Go e nmen (CGIC10/172),
Spanish Minis y o Educa ion (G an CTQ2017-84415-R, MAT2015-69967-C3-1R),
he Gene ali a de Ca alunya (2014/SGR/
and he Ins i u o de Salud Ca los III (CB06_01_0019, CB06_01_1028). The
au ho s also wish o hank he in ellec ual and echnical assis ance om he ICTS
“NANBIOSIS”, mo e speci ically by he D ug Fo mula ion Uni (U10) o he CIBER
in Bioenginee ing, Bioma e ials, and Nanomedicine (CIBER-BBN) a he Uni e si y
o Basque Coun y (UPV/EHU). Technical and human suppo p o ided by SGIke
(UPV/EHU) is acknowledged.
5.7. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://
doi.o g/10.1016/j.jcon el.2019.05.010.
Chap e 5
123
Supplemen a y ig.1. (A) ARPE-19 cell iabili y a e 4h o incuba ion wi h di e en
concen a ions o chlo oquine diphospha e. Phase con as mic oscopy o ARPE-19
cells showing con ol cells (C l) and cells wi h acuola ed cy oplasm (CQ) a e hei
ea men wi h chlo oquine diphospha e 100ug/ml.
Supplemen a y ig. 2. Con ocal luo escence mic og aphs o (A) e inal whole moun
a e 3 days o in a i eal adminis a ion o DPP80 nioplexes and o (B) e inal c oss-
sec ions a e 3 days o sub e inal adminis a ion o DPP80 nioplexes. The e is no GFP
exp ession de ec ed in di e en e inal laye s. Scale ba s: 20 μm.
Chap e 6
130

Chap e 6
131
Chap e 6
Gene al discussion
Chap e 6
132
Chap e 6
133
P esen ly, ca ionic niosomes, as non- i al gene deli e y ca ie , ha e become
an impo an ool o deli e bo h gene ic mac omolecules and d ug molecules.
De elopmen o e icien non- i al gene deli e y sys ems could educe he ime and
sa e expenses o coming new ma ke he apies. In addi ion, hey would be
signi ican ly sa e han hei i al coun e pa s. De elopmen o sa e and e icien non-
i al ec o s o deli e DNA in o he CNS ep esen s a huge challenge o ace many
neu ological diso de s. Despi e i s pe iphe al loca ion, he e ina o neu al laye o he
eye, is ac ually a pa o he cen al ne ous sys em.
In he cu en s udy, we ha e designed, p epa ed and cha ac e ized niosome
o mula ions based on di e en ca ionic lipids and a ious helpe molecules. In
i o s udies we e conduc ed o e alua e ans ec ion e iciency, iabili y and
in e naliza ion mechanism in ARPE-19 and NT2 cells. Subsequen ly, hei in i o
applica ion was e alua ed in bo h e ina and b ain.
6.1 Lycopene enhances he e icacy o ca ionic niosomes based on DOTMA and
polyso ba e 60 o e inal gene deli e y pu poses
Due o i s appealing chemical s uc u e, he comme cially a ailable ca ionic lipid
DOTMA has been used widely o gene deli e y applica ions (1).
Fig. 1. Chemical s uc u es o he ca ionic lipid, N-[1-(2,3-dioleoyloxy)p opyl]-N,N,N-
ime hylammonium chlo ide (DOTMA) (A), Polyso ba e 60 (B), and Lycopene (C).
Chap e 6
134
As shown in Fig. 1-A, i s s uc u e is composed o a pola head-g oup, wo non-pola
hyd ophobic chains, a linke and a back-bone, which classically a e known as he ou
domains ha ule gene ans ec ion p ocess (2).
We combined DOTMA wi h he non-ionic su ac an polyso ba e 60, in a
niosome o mula ion a a mola a io o 1:4 espec i ely, in o de o enhance cell
ole ance (3) and p o ide a s e ic ba ie o a oid agg ega ion (4). I has been epo ed
on he li e a u e ha he p esence o PEG chains in he chemical s uc u e o
polyso ba es (Fig. 1-B) p o ides physicochemical s abili y o lipid o mula ions (5),
conse es effec i eness o e ime and boos s ans ec ion efficiency (6).
Table 1
Physical cha ac e iza ion o DP60 and DP60L niosomes ega ding pa icle size (nm);
Polydispe si y index (PDI), and Ze a po en ial (mV). Da a ep esen mean ± SD (n =
3).
Pa icle
Size (nm)
PDI
Ze a po en ial (mV)
DP60 niosome
66.49 ± 1.17
0.46±0.02
45.30±1.57
DP60L niosome
101.60 ± 2.48
0.44±0.02
33.80±1.13
Compa ed wi h polyso ba e 80, ano he polyso ba e ha has been widely used in he
elabo a ion o niosome o mula ions o gene deli e y applica ions, [15–17,31]
polyso ba e 60 could offe some impo an ad ances.
Chap e 6
135
Fig. 2. Physicochemical cha ac e iza ion o nioplexes. A) Effec o ca ionic lipid/DNA
mass a io (w/w) on bo h pa icle size (ba s) and ze a po en ial (lines). Each da a poin
ep esen s he mean ± SD (n = 3). TEM o DP60 (B1) and DP60L nioplexes (B2) a
a io o 18/1 ca ionic lipid/DNA mass a io (w/w). Scale ba = 500 nm. Binding, SDS-
induced elease and p o ec ion o DNA a diffe en ca ionic lipid/DNA mass a ios
(w/w) o nioplexes based on bo h DP60 (C1) and DP60L (C2) isualized by aga ose
elec opho esis. Lanes 1–3 co espond o uncomplexed DNA; lanes 4–6, ca ionic
lipid/DNA mass a io 6/1; lanes 7–9, ca ionic lipid/DNA mass a io 12/1; lanes 10–12,
ca ionic lipid/DNA mass a io 18/1; lanes 13–15, ca ionic lipid/DNA mass a io 22/1.
Nioplexes we e ea ed wi h SDS (lanes 2, 5, 8, 11 and 14) and DNase I + SDS (lanes
3, 6, 9, 12 and 15). OC: open ci cula o m, SC: supe coiled o m.
Fo ins ance, he lack o double bonds in he hyd oca bon chains (Fig. 1-B) could
p o ide low pe meabili y o he esicles, and he e o e be e s abili y o niosome
memb anes (5). Addi ionally, compa ed wi h o he hyd ophilic su ac an s such as
polyso ba es 80, 40 o 20, he low hyd ophilic-lipophilic balance (HLB) alue o
polyso ba e 60 (14.9) could help o solubilize lycopene mo e efficien ly (7). The
addi ion o he na u al and non-pola lipid lycopene (Fig. 1-C) in o niosome bilamina
memb ane could inc ease i s luidi y, dis u b memb ane packing, and consequen ly
esicle suscep ibili y o en i onmen al s esses (8).

Chap e 6
136
Fig. 3. In i o ans ec ion efficiency and cell iabili y in ARPE-19 cells a 72 h pos -
ans ec ion. (A) Flow cy ome y-based e alua ion o he pe cen age o EGFP-posi i e
cells (ba s) and pe cen age o iable cells (lines) a diffe en ca ionic lipid/DNA mass
a ios (w/w). Values ep esen mean ± SD (n = 3). (*P < 0.05 s. Lipo ec amine™2000
ans ec ion). (#P < 0.05 s. Lipo ec amine™2000 iabili y). (B) O e lay o
luo escence and phase-con as mic og aphs o ARPE-19 cells 72 h pos - ans ec ion
a diffe en ca ionic lipid/DNA mass a ios (w/w). Scale ba = 100 μm.
Once elabo a ed by he e e se phase elabo a ion me hod, bo h DP60 and DP60L
niosomes showed app op ia e size (in he nanome ic scale) and PDI alues (below 0.5)
o gene deli e y pu poses (Table 1). High posi i e ZP alues (> +25 mV) ensu e long-
las ing s abili y (9), ex empo e elec os a ic ecip ocal ac ion wi h DNA, along wi h
binding o he nioplexes o he nega i ely cha ged uni s o he cell memb ane p e ious
o cellula up ake (2).
Chap e 6
137
Fig. 4. Up ake o FITC-labeled nioplexes in ARPE-19 cells. Bo h DP60 and DP60L a
a mass a io o 18/1 (w/w). (A) Pe cen age o FITC-posi i e cells. Da a ep esen mean
± SD (n = 3). *p < 0.05. (B) Fluo escence mic og aphs o ARPE-19 cells a 2 h and 4
h o incuba ion wi h FITC-labeled DP60 and DP60L nioplexes (g een). Nuclei s ained
wi h Dapi (blue). O iginal magni ica ion 63×. Scale ba = 20 μm. (Fo in e -p e a ion
o he e e ences o colo in his igu e legend, he eade is e e ed o he web e sion
o his a icle.)
To elabo a e nioplexes, we added pCMS-EGFP epo e plasmid o bo h niosome
o mula ions a diffe en ca ionic lipid/DNA mass a ios, since o he wise, he complex
assembly p ocess could be slowed down (10). The sligh changes disce ned in he size
o nioplexes (100–150 nm, Fig. 2-A), a he mass a ios s udied, migh be due o he
delica e balance o diffe en e en s in ol ed in he mul is ep sel -assembled complex
o ma ion, such as: elec os a ic in e ac ion, u he memb ane me ging, lipid mixing
and agg ega e g ow h (10). Rega ding he ZP alues, he g adual inc ease o supe icial
cha ge along wi h ca ionic lipid/DNA a ios (w/w) sugges s he capaci y o ca ionic
Chap e 6
138
niosomes o bind o and neu alize he nega i ely cha ged phospha e g oups o DNA
(11). Lycopene addi ion educed ZP alue o DP60L nioplexes, compa ed o DP60, a
all a ios s udied. This ac could be explained by he pe u ba ion o he lipid memb ane
bilaye , which could dissipa e he elec ical po en ial (Fig. 2-A) (12).
Fig. 5. Th ee-channel o e lay RGB images o ARPE-19 cells showing nioplexes wi h
FITC-labeled pCMS-EGFP (g een) and one o he endocy osis ma ke s in ed
(AlexaFluo ® 555-Chole a Toxin, AlexaFluo ® 546-T ans e in o AlexaFluo ® 594-
dex an). P esence o yellow/o ange colo ep esen s he o e lay o an endocy ic ma ke
and nioplexes. (M = Mande 's o e lap coefficien ). O iginal magni ica ion 63×, Scale
ba = 25 μm. (Fo in e p e a ion o he e e ences o colo in his igu e legend, he
eade is e e ed o he web e sion o his a icle.)
In any case, bo h o mula ions could unc ion as gene deli e y ca ie s, since
complexes wi h posi i e cha ge could in e ac elec os a ically wi h he anionic cell
coa , inducing ea ly s eps o he endocy osis p ocess (13). The high posi i e ZP alue
o bo h DP60 and DP60L nioplexes, especially a 18/1 mass a io (42 and 27 mV,
Chap e 6
139
espec i ely), could ensu e he disc e e mo phology and absence o agg ega es
obse ed by TEM mic og aphs (Fig. 2-B) (14). Among o he ac o s ha can command
ans ec ion success, he an de Waals in e ac ions be ween phospha e g oups o he
DNA (nega i ely cha ged) and amine g oups o he ca ionic niosomes (posi i ely
cha ged) dese e special a en ion (11, 15, 16). We obse ed by aga ose gel
elec opho esis assay ha a all ca ionic lipid/DNA a ios es ed, bo h niosomes we e
capable o condense, elease and p o ec he DNA om enzyma ic diges ion (Fig. 2-
C1and C2).
Once we e alua ed ha ou nano- o mula ions we e bio echnologically i ing
o gene deli e y pu poses, we p oceeded o e alua e hei biological pe o mance in
ARPE-19 cells.
ARPE-19 cell line has a no mal ka yo ype and has unc ional and s uc u al p ope ies
simila o e inal pigmen epi helia (RPE) in i o, exp esses RPE-speci ic ma ke s,
hence i is conside ed a sui able ans ec ion model o in es iga e ou ec o s'
effec i eness and sa e y be o e i s applica ion in i o (17). I has been epo ed ha he
non-ionic na u e o su ac an s makes niosomes well ole a ed by cells (18). Ou esul s
in Fig. 3 show highe cell iabili y alues in cells ans ec ed wi h bo h nioplexes when
compa ed wi h cells ans ec ed wi h Lipo ec amine™ 2000. Addi ionally, we obse ed
unde he luo escence mic oscope ha cells ans ec ed wi h bo h nioplexes
main ained hei no mal mo phology, e en a high ca ionic lipid/DNA a ios (Fig. 3-
B). Al hough he pe cen age o ans ec ed cells wi h DP60L niosomes a 18/1 mass
a io was signi ican ly lowe han ha ob ained wi h comme cially a ailable
Lipo ec amine™ 2000, ou niosomes o mula ion was be e ole a ed by ARPE-19
cells. The e o e, i could be an in e es ing al e na i e o Lipo ec amine™ 2000, since
some au ho s ha e epo ed damage on he e ina associa ed o he in i o
adminis a ion o Lipo ec amine™ 2000 in he eye (19). Rega ding he ans ec ion
efficiency, he lipid composi ion is conside ed a p ima y limi ing ac o ha affec s o
his p ocess (1). We clea ly obse ed in Fig. 3 he impac ha lycopene had on
ans ec ion efficiency in ARPE-19 cells, since alues we e clea ly highe when
lycopene was p esen in he niosome o mula ion.