In e na ional Jou nal o Pha maceu ics 639 (2023) 122968
A ailable online 18 Ap il 2023
0378-5173/© 2023 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
Long- e m biophysical s abili y o nanodiamonds combined wi h lipid
nanoca ie s o non- i al gene deli e y o he e ina
Nuseibah H. AL Q aish
a
,
b
,
1
, Ilia Villa e-Bei ia
a
,
b
,
c
,
1
, Idoia Gallego
a
,
b
,
c
, Gema Ma ínez-
Na a e e
b
,
, C is ina So o-S´
anchez
b
,
, My iam Sainz-Ramos
a
,
b
,
c
, Tania B Lopez-Mendez
a
,
b
,
c
,
Alejand o J. Pa edes
d
,
e
, F ancisco Ja ie Chich´
on
g
, Noelia Zama e˜
no
g
, Edua do Fe n´
andez
b
,
,
Gus a o Pu as
a
,
b
,
c
,
*
, Jos´
e Luis Ped az
a
,
b
,
c
,
*
a
NanoBioCel Resea ch G oup, Labo a o y o Pha macy and Pha maceu ical Technology. Facul y 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
Ne wo king Resea ch Cen e o Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN), Ins i u e o Heal h Ca los III, Paseo de la Uni e sidad, 7, 01006 Vi o ia-
Gas eiz, Spain
c
Bioa aba, NanoBioCel Resea ch G oup, 01009 Vi o ia-Gas eiz, Spain
d
Resea ch and De elopmen Uni in Pha maceu ical Technology (UNITEFA), CONICET and Depa men o Pha maceu ical Sciences, Chemis y Sciences Facul y,
Na ional Uni e si y o C´
o doba, Haya de la To e y Medina Allende, X5000XHUA C´
o doba, A gen ina
e
School o Pha macy, Queen’s Uni e si y Bel as , Medical Biology Cen e, 97, Lisbu n Road, Bel as , BT9 7BL No he n I eland, UK
Neu op o hesis and Neu oenginee ing Resea ch G oup, Ins i u e o Bioenginee ing, Miguel He n´
andez Uni e si y, A enida de la Uni e sidad, 03202 Elche, Spain
g
C yoEM CSIC Facili y. Cen o Nacional de Bio ecnología (CNB-CSIC). S uc u e o Mac omolecules Depa men . Calle Da win n◦3, 28049 Mad id, Spain
ARTICLE INFO
Keywo ds:
Nanodiamond
Niosome
Non- i al
Gene deli e y
S abili y
Re ina
ABSTRACT
Nanodiamonds we e combined wi h niosome, and esul ing o mula ions we e named as nanodiasomes, which
we e e alua ed in e ms o physicochemical ea u es, cellula in e naliza ion, cell iabili y and ans ec ion e -
iciency bo h in in i o and in in i o condi ions. Such pa ame e s we e analyzed a 4 and 25
◦
C, and a 15 and 30
days a e hei elabo a ion. Nanodiasomes showed a pa icle size o 128 nm ha was main ained o e ime
inside he ±10% o de ia ion, unless a e 30 days o s o age a 25 ◦C. Some hing simila occu ed wi h he
ini ial ze a po en ial alue, 35.2 mV, being bo h o mula ions mo e s able a 4 ◦C. The inco po a ion o nano-
diamonds in o niosomes esul ed in a 4- old inc ease o ans ec ion e iciency ha was main ained o e ime a 4
and 25 ◦C. In i o s udies epo ed high ansgene exp ession o nanodiasomes a e sub e inal and in a i eal
adminis a ion in mice, when injec ed eshly p epa ed and a e 30 days o s o age a 4 ◦C.
1. In oduc ion
The pha maceu ical science has di ec ed conside able e o s o-
wa ds disco e ing and de eloping sa e and e icien ec o s o gene
he apy pu poses. While mos s udies ocus on o e coming speci ic is-
sues ela ed o con en ional gene deli e y pla o ms, such as unp e-
dic abili y, incompa ibili y wi h biological sys ems o low e iciency,
ew s udies conduc an exhaus i e assessmen o he s o age s abili y o
gene ca ie s, a c i ical quali y o achie e bo h la ge-scale p oduc ion
and clinical applica ion (Suzuki e al., 2015).
Nowadays, ew gene he apy d ugs ha e been ma ke ed globally, and
mos o hese p oduc s a e based on i al ec o s (Al Q aish e al., 2020;
Shah ya i e al., 2019). Howe e , because o speci ic issues associa ed o
i al gene ca ie s, including low DNA packing capaci y, high cos s and
complex p oduc ion, non- i al ec o s a e gaining inc easing in e es
(Do e al., 2019; Ib aheem e al., 2014; Ginn e al., 2018). In addi ion o
o e coming hese speci ic challenges, non- i al ec o s o e high
e sa ili y due o he wide a ie y o a ailable nanoma e ials ha can be
used o p oduce gene deli e y sys ems (G ijal o e al., 2019; Riley and
Ve me is, 2017). Among hese, niosomes ha e been epo ed in
epea ed occasions as e icien ehicles o gene deli e y o b ain
(Mashal e al., 2018) and e ina (Pu as e al., 2015), among o he s.
* Co esponding au ho s a : NanoBioCel Resea ch G oup, Labo a o y o Pha macy and Pha maceu ical Technology. Facul y 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.
E-mail add esses: [email p o ec ed] (G. Pu as), [email p o ec ed] (J.L. Ped az).
1
These au ho s con ibu ed equally o his wo k.
Con en s lis s a ailable a ScienceDi ec
In e na ional Jou nal o Pha maceu ics
jou nal homepage: www.else ie .com/loca e/ijpha m
h ps://doi.o g/10.1016/j.ijpha m.2023.122968
Recei ed 9 Decembe 2022; Recei ed in e ised o m 6 Ma ch 2023; Accep ed 14 Ap il 2023
In e na ional Jou nal o Pha maceu ics 639 (2023) 122968
2
Niosomes a e ca ionic lipid nanopa icles wi h a bilaye dis ibu ion
simila o liposomes, bu , addi ionally, niosomes can also con ain a
“helpe ” componen and a non-ionic su ac an o ob ain mo e s able
colloidal dispe sions. All hese men ioned componen s, p o ide nio-
somes supe io chemical and s o age s abili y han liposomes (Ba elds
e al., 2018; Ojeda e al., 2016). All he componen s o niosome o -
mula ions in luence on hei biocompa ibili y and ans ec ion e i-
ciency. In pa icula , he cha ac e is ics o he “helpe ” componen
in luence di ec ly on ele an biological p ocesses, such as he cellula
up ake and he subsequen in acellula disposi ion, which a e c i ical
ac o s ha de e mine success ul gene deli e y e iciency (Ojeda e al.,
2016). Among he mos s udied “helpe ” componen s, lipid-based ones
such as lycopene, choles e ol, squalane, squalene and sphingolipids (Al
Q aish e al., 2021; Mashal e al., 2017; Ojeda e al., 2016) ha e been he
mos employed o da e, bu also non-lipid ones such as chlo oquine a e
gaining in e es wi h encou aging esul s (Mashal e al., 2019).
Recen ly, nanodiamonds (NDs) ha e eme ged as an in e es ing ma-
e ial o elabo a e non- i al ec o s o gene deli e y applica ions. The
high biocompa ibili y, low oxici y, along wi h hei e sa ile su ace
chemis y (Lim e al., 2016), which allows mul iple combina ion o ms
as “helpe ” componen s wi h o he nanoma e ials such as polyme s o
lipids ha e cap u ed he in e es o scien i ics. NDs a e allo opes o
ca bon ha con ain a co e diamond c ys alline s uc u e and p esen
unique physicochemical p ope ies, such as almos sphe ical shape, low
size polydispe si y and high speci ic a ea. Addi ionally, NDs can be
easily unc ionalized wi h many chemical compounds (Chauhan e al.,
2020). In p e ious esea ch o gene he apy pu poses, au ho s com-
bined NDs wi h hyd ophilic ca ionic polyme s such as polye hylenimine
800 (PEI 800) (Alhaddad e al., 2011); Chen e al., 2021; Zhang e al.,
2009) and polyallylamine hyd ochlo ide (PAH) (Alhaddad e al., 2011),
o wi h ca ionic monome such as lysine (Alwani e al., 2016) by elec-
os a ic in e ac ions. On he o he hand, co alen de i a iza ion o NDs
has been pe o med wi h silane-NH
2
g oups (Edging on e al., 2018;
Zhang e al., 2009) and polyamidoamine (PANAM) (Lim e al., 2017). In
o he s udy, Bi e al designed and syn he ized a complex s uc u e o ND-
CONH(CH
2
)
2
NH-VDGR/su i in-siRNA wi h an i umo al e ec (Bi
e al., 2016). Finally, ou esea ch g oup combined NDs wi h niosomes,
demons a ing hei supe io i y in enhancing he ans ec ion e iciency
o hese non- i al ec o s (Al Q aish e al., 2022). Howe e , o he bes o
ou knowledge, he combina ion o NDs wi h niosomes o e alua e hei
s abili y along wi h hei e inal gene deli e y e iciency has no been
explo ed ye .
In his wo k, we p epa ed and compa a i ely e alua ed he ans-
ec ion e iciency and long- e m s abili y a di e en s o age empe a-
u es o wo niosome-based o mula ions ha only di e ed on he use o
no o NDs as “helpe ” componen s. Fo mula ions we e based on ca ionic
lipid N-[1-(2,3dioleoyloxy)p opyl]-N,N,N- ime hylammonium chlo-
ide (DOTMA) and non-ionic su ac an polyso ba e 20. NDs we e added
as “helpe ” componen s o one o he wo o mula ions. Resul ing o -
mula ions we e named as niosomes and nanodiasomes depending on
hei ND con en and we e incuba ed wi h pCMS-EGFP plasmid in o de
o ob ain nanocomplexes, named as nioplexes and nanodiaplexes,
espec i ely. Fo mula ions we e e alua ed in e ms o physicochemical
p ope ies, including size dis ibu ion, supe icial cha ge and poly-
dispe si y index a di e en pe iods o ime (0, 15 and 30 days) and
s o age empe a u es (4 ◦C and 25 ◦C). In addi ion, in i o biological
s udies we e pe o med o e alua e he oxici y o he o mula ions
along wi h hei cellula up ake and gene deli e y e iciency o e ime a
di e en s o age empe a u es in HEK-293 cells. Fu he assays we e
ca ied ou in p ima y e inal cells and in mice a e bo h in a i eal
and sub e inal adminis a ion o he o mula ions in o de o de e mine
he e ec o NDs as “helpe ” componen s on he gene deli e y e iciency
and long- e m s o age o he o mula ions.
2. Ma e ials and me hods
2.1. P epa a ion o nanodiasome and niosome o mula ions
Fo he p epa a ion o he nanodiasome o mula ions, he wa e in
oil emulsion echnique was used as p e iously desc ibed. B ie ly. 250
μ
L
o NDs (10 mg/ml in H
2
O, Sigma-Ald ich Mad id, Spain, p oduc ID:
900180) we e ul asonica ed o 30 min and mixed wi h an aqueous
phase composed o 2 mL o 0.5% polyso ba e 20 (Sigma-Ald ich Mad id,
Spain) plus 1.75 mL o MilliQ wa e . On he o he hand, 5 mg o DOTMA
(A an i Pola Lipids, Inc., Alabama, USA) we e accu a ely weigh ed and
dilu ed in 1 mL o he o ganic sol en dichlo ome hane (DCM) (Pan eac,
Ba celona). This oil phase was inco po a ed in o he aqueous phase and
sonica ed o 30 s a 50 W (B anson Soni ie 250, Danbu y). The
emulsion was main ained unde magne ic s i ing o 2 h a oom em-
pe a u e (RT) un il e apo a ion o DCM o ob ain he nanodiasome
o mula ion. The p epa a ion o he niosome o mula ion ollowed he
same p ocedu e, bu he aqueous phase did no con ain NDs. Fig. 1
summa izes he main componen s and hei disposi ion in bo h
o mula ions.
2.2. P epa a ion o he nanocomplexes
Nanocomplexes we e ob ained by incuba ing bo h niosomes and
nanodiasomes wi h he p e iously p opaga ed pEGFP plasmid, as
desc ibed elsewhe e (Ojeda e al., 2016), o ob ain complexes (nioplexes
and nanodiaplexes, espec i ely) a 5/1 ca ionic lipid/DNA a io (w/w).
2.3. Physicochemical s udies
Niosomes, nanodiasomes, and hei co esponding complexes we e
physicochemically cha ac e ized by means o mean pa icle size, dis-
pe si y index (ᴆ) and ze a po en ial, ollowing p e iously epo ed
me hodology (Mashal e al., 2017).
Mic oscopy s udies we e ca ied ou o de e mine he mo phology
and he disposi ion o NDs in he nanodiasomes, by c yo-elec on
omog am, as p e iously desc ibed (Al Q aish e al., 2022).
2.4. Biophysical s abili y s udies o o mula ions
S abili y s udies we e pe o med wi h all o mula ions by means o
physicochemical cha ac e iza ion and biological pe o mance. Fo ha
pu pose, pa icle size, dispe si y, ze a po en ial, cell iabili y and
ans ec ion we e e alua ed a 0, 15 and 30 days wi h s o ed o mula-
ions a 4 ◦C and 25 ◦C. Cellula up ake, ans ec ion in p ima y e inal
cell cul u es, along wi h in i o e inal assays we e pe o med wi h
eshly p epa ed o mula ions and wi h o mula ions s o ed a 4 ◦C o
30 days.
2.5. T ans ec ion s udies
To pe o m ans ec ion expe imen s, human emb yonic kidney 293
cell line (HEK-293; ATCC® CRL1573
TM
) was cul u ed and main ained as
p e iously desc ibed (Ojeda e al., 2016). Fo his, HEK-293 cells we e
seeded a 20 ×10
4
cells pe well in 24 well pla es and incuba ed o each
70% o con luence he nex day. A e disca ding he medium om he
wells, cells we e ans ec ed using Op iMEM (Gibco, San Diego, CA,
USA) ans ec ion medium, o 4 h wi h nioplexes and nanodiaplexes,
om eshly p epa ed and s o ed o 15 and 30 days a 4 ◦C and 25 ◦C
o mula ions, a he ca ionic lipid/DNA mass a io 5/1, as p e iously
epo ed (Al Q aish e al., 2022). Posi i e con ol o ans ec ion con-
sis ed in cells ans ec ed wi h Lipo ec amine 2000™ (In i ogen,
Ca lsbad, CA, USA), while nega i e con ol we e non- ea ed cells bu in
Op iMEM o 4 h. Each condi ion was ca ied ou in iplica e.
N.H. AL Q aish e al.
In e na ional Jou nal o Pha maceu ics 639 (2023) 122968
3
2.6. EGFP exp ession and cell iabili y assays
The e iciency o he ans ec ion p ocess was assessed bo h quali-
a i e and quan i a i ely 48 h a e he ans ec ion assay. Quali a i e
de e mina ion o EGFP signal was pe o med using an in e ed luo-
escence mic oscope (Eclipse TE2000-S, Nikon). Quan i a i e s udies o
plasmid exp ession, cell iabili y and mean luo escence in ensi y (MFI)
we e ca ied ou by low cy ome y using a FACSCalibu sys em (Bec on
Dickinson Bioscience, San Jose, USA), as epo ed p e iously (Al Q aish
e al., 2022).
2.7. Cellula up ake
To analyse he cellula in e naliza ion p ocess o nioplexes and
nanodiaplexes, using eshly p epa ed and s o ed o 30 days a 4 ◦C
o mula ions, niosomes and nanodiasomes we e condensed wi h FITC-
labelled pEGFP plasmid. Fluo escence mic oscopy and low cy ome y
equipmen we e used o elabo a e cellula up ake p ocess in a quali a-
i e and quan i a i e way, espec i ely (Al Q aish e al., 2022).
2.8. Animals and anes he ics
P ocedu es we e pe o med ollowing he RD 53/2013 Spanish and
2010/63/EU Eu opean Union egula ions, as well as he Associa ion o
Resea ch in Vision and Oph halmology (ARVO), once ob ained he
app o al o he Miguel He nandez Uni e si y S anding Commi ee o
Animal Use in he Labo a o y.
2.9. T ans ec ion s udies in a p ima y cen al ne ous sys em cell
cul u es and immunocy ochemis y assays
E17-E18 a emb yos (Sp ague Dawley) we e employed o he
ex ac ion o p ima y cen al ne ous sys em (CNS) cells, om he b ain
co ex and e inal issue. Cells we e emo ed and cul u ed on o p e-
coa ed glass co e slips in 24 well pla es.
Co ical and e inal cells we e ans ec ed wi h nanodiaplexes om
eshly p epa ed and 30 days s o ed nanodiasomes. Lipo ec amine™
2000 (The moFishe Scien i ic) was used as a posi i e con ol. T ans-
ec ions expe imen s we e epea ed h ee imes o each condi ion and
GFP exp ession was analyzed a 96 h a e ans ec ion.
Cell ixa ion was ca ied ou wi h 4% pa a o maldehyde o 25 min
and pe meabilized using 0.5% T i on X-100 du ing 5 min. A e blocking
wi h a solu ion o 10% BSA ( / ) in PBS o 1 h a RT, cells we e
incuba ed wi h p ima y an ibody chicken an i-EGFP (The moFishe
Scien i ic) o e nigh a 4 ◦C. Seconda y an ibody Alexa Fluo 555 goa
an i-chicken IgG (The moFishe Scien i ic) and Hoechs 33,342 (Sigma-
Ald ich, Spain) we e applied o 1 h a 4 ◦C. Co e slips we e analyzed
by a Zeiss AxioObse e Z1 (Ca l Zeiss) mic oscope equipped wi h an
ApoTome sys em and Leica TCS SPE spec al con ocal mic oscope (Leica
Mic osys ems GmbH, We zla , Ge many).
2.10. In a i eal and sub e inal adminis a ion o o mula ions
In i o ans ec ions o nanodiaplexes we e ca ied in C57BL/6J mice
wi h eshly p epa ed (n =10) and 30 days s o ed nanodiasomes (n =
10). Animals we e anes he ized, and in a i eal (n =5) o sub e inal (n
=5) injec ions we e adminis e ed unde mic oscope (Zeiss OPMI® pico;
Ca l Zeiss Medi ec GmbH, Jena, Ge many) using a Hamil on mic o-
sy inge wi h a blun 34-gauge needle (Hamil on Co., Reno, NV). The
nanodiaplexes solu ion injec ed was 0.5
μ
L which con ained 100 ng o
EGFP plasmid. As nega i e con ols, he un ea ed igh eyes we e used.
EGFP exp ession was analyzed quali a i ely one week a e he in-
jec ion o complexes om eshly o 30 days s o ed nanodiasomes in
ozen sec ions o he e ina, as p e iously desc ibed (Mashal e al.,
2017). C yosec ions we e incuba ed wi h he p ima y an ibodies
chicken an i-EGFP (The moFishe Scien i ic) and abbi an i-Iba1
(Abcam) o e nigh a 4 ◦C. Seconda y an ibodies Alexa Fluo 488
donkey an i- abbi and Alexa Fluo 555 goa an i-chicken (bo h The -
moFishe Scien i ic) we e applied o 1 h a 4 ◦C. Nuclei we e s ained
wi h Hoechs 33,342 (The mo Fishe Scien i ic). The samples we e
analyzed and pho og aphed using a Leica TCS SPE spec al con ocal
mic oscope (Leica Mic osys ems GmbH, We zla , Ge many).
2.11. S a is ical analysis
Da a we e analyzed using SPSS 15.0 so wa e. No mali y and ho-
mogenei y o a iances we e e alua ed wi h he Shapi o-Wilk es and
he Le ene es , espec i ely. S uden s es o ANOVA ollowed by pos -
Fig. 1. O e iew o o mula ions and hei componen s.
N.H. AL Q aish e al.
In e na ional Jou nal o Pha maceu ics 639 (2023) 122968
4
hoc HSD Tukey es we e employed unde pa ame ic condi ions. On he
con a y, K uskal-Wallis es and/o Mann-Whi ney U es we e used
unde non-pa ame ic condi ions. In all cases, P alue ≤0.05 was
conside ed s a is ically signi ican . Da a we e ep esen ed as mean ±
s anda d de ia ion (SD).
3. Resul s
3.1. Physicochemical cha ac e iza ion o o mula ions
In gene al, o mula ions con aining NDs p esen ed highe mean
pa icle size alues han hei coun e pa s in all condi ions (Fig. 2A). A
day 0, eshly p epa ed nanodiasome o mula ions showed a mean
pa icle size o 128.7 ±4.2 nm, which main ained s able o e ime and
was signi ican ly inc eased (P <0.05) only a e 30 days o s o age a
25 ◦C. Rega ding he niosome o mula ion, he mean pa icle size a day
0 was 90.5 ±10.3 nm and p esen ed signi ican oscilla ions (P <0.05) a
day 15 o s o age a 25 ◦C and a day 30 o s o age a 4 ◦C.
The measu emen o ze a po en ial o nanodiasomes and niosomes a
di e en days and empe a u es o s o age e ealed mo e oscilla ions in
he case o niosome o mula ions han hei coun e pa s (Fig. 2B). The
mean ze a po en ial alue o eshly p epa ed nanodiasomes a day
0 was 35.2 ±0.3 mV and p esen ed a s a is ically ele an inc ease (P <
0.05) a e 30 days o s o age a bo h 4 ◦C and 25 ◦C. On he o he hand,
niosome o mula ions showed a mean ze a po en ial alue o 20.2 ±2.5
mV a day 0, which signi ican ly inc eased a e 15 days o s o age a
25 ◦C (P <0.05) and dec eased a e 30 days o s o age a 4 ◦C (P <0.05)
and 25 ◦C (P <0.01).
Dispe si y alues o nanodiasomes we e lowe han niosomes and
emained s able wi h li le oscilla ions a all condi ions es ed, while
niosome o mula ions showed highe alues and mo e a ia ions,
especially a e being s o ed du ing 15 days a 25 ◦C (Fig. 2C).
Nanodiasomes unde elec on c yo- omog aphy mic oscopy
(Fig. 2D) showed a sphe ical shape, wi h he NDs in eg a ed in he lipid
laye (Fig. 2D, whi e a ow).
3.2. Gene deli e y e iciency and oxici y o nioplexes and nanodiaplexes
The compa a i e e alua ion o cell iabili y and gene deli e y e i-
ciency in cells be ween nanodiaplexes and nioplexes, p epa ed wi h
esh o mula ions o wi h o mula ions s o ed o 15 and 30 days a
di e en empe a u es, showed ha nanodiaplexes we e be e ole a ed
by cells and achie ed signi ican ly highe ans ec ion a es a all con-
di ions. Da a we e no malized o Lipo ec amine 2000
TM
which epo ed
39.5 ±12.2% o EGFP exp ession in li e cells, and 79.7 ±14.6% cell
iabili y in HEK-293 cells (da a no shown). The mean pe cen age o li e
cells exposed o eshly p epa ed nanodiaplexes was 90.79 ±2.5%,
while his alue was signi ican ly lowe (P <0.001) o nioplexes which
p esen ed a mean pe cen age o li e cells o 78.8 ±5.8% (Fig. 3A, lines).
These alues emained ela i ely s able o e ime and di e en s o age
empe a u es, wi h li le oscilla ions bu no s a is ically ele an di e -
ences compa ed o he alues o day 0 in bo h o mula ions.
Rega ding ans ec ion e iciency, he pe cen age o EGFP exp essing
li e cells exposed o eshly p epa ed nanodiaplexes and nioplexes we e,
espec i ely, 89.8 ±3.4% and 23.3 ±1.1% (Fig. 3A, ba s). These alues
emained s able o bo h o mula ions o e ime and s o age condi ions,
always main aining signi ican ly highe ans ec ion pe cen ages in cells
ea ed wi h nanodiaplexes han wi h nioplexes (P <0.001). In addi ion,
he MFI da a (Fig. 3B) co obo a ed he ad an age o nanodiaplexes o e
nioplexes, wi h signi ican ly highe MFI alues ob ained in cells exposed
o nanodiaplexes p epa ed wi h nanodiasome o mula ions a all days
and s o age condi ions es ed (P <0.001). Fig. 3C and D show ep e-
sen a i e luo escence mic oscopy images o HEK-239 ans ec ed cells
wi h bo h o mula ions a day 0 and a e 30 days o s o age a 4 ◦C,
espec i ely.
Fig. 2. Physicochemical cha ac e iza ion and s abili y o o mula ions a di e en days and s o age empe a u e. A. Mean pa icle size. B. Ze a po en ial. C. Dis-
pe si y. Each alue shows he mean ±SD o 3 eadings. Blue and o ange s ipes ep esen ±10% de ia ion espec o nanodiasomes and niosomes pa ame e s a day
0, espec i ely. D. C yo-elec on omog am slice o a nanodiasome; as e isk indica es he aqueous phase; whi e a ow indica es he lipid laye o he nanodiasome
wi h nanodiamonds in eg a ed in he lipid s uc u e; black a ow indica es highe densi ies o he omog am (mo e elec on-dense ma e ial), which co espond o
gold nanopa icles added o he sample o il se ies alignmen . Scale ba : 100 nm. (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.)
N.H. AL Q aish e al.
In e na ional Jou nal o Pha maceu ics 639 (2023) 122968
5
3.3. Cellula up ake o nioplexes and nanodiaplexes
The analysis o cellula up ake in HEK-293 cells 4 h a e exposu e o
complexes p epa ed wi h esh and 30 days a 4 ◦C s o ed nanodiasomes
and niosomes, e ealed signi ican ly highe (P <0.05) cell in e naliza-
ion pe cen ages o ND based o mula ions, a bo h condi ions (Fig. 4A).
These cell up ake pe cen ages emained s able o e ime o bo h o -
mula ions, wi h s a is ically ele an di e ences. Such alues we e
no malized o Lipo ec amine 2000
TM
which epo ed 43.5 ±2.7% o
FITC-pEGFP posi i e cells 4 h a e ans ec ion (da a no shown).
Fig. 4B shows ep esen a i e images o cellula up ake in HEK-293 cells
exposed o bo h o mula ions a days 0 and 30.
3.4. Gene deli e y e iciency o nanodiaplexes in a p ima y cell cul u es
The ans ec ion assay o nanodiaplexes in a p ima y e inal cells
wi h eshly p epa ed (Fig. 5A) and 30 days s o ed nanodiasomes a 4 ◦C
(Fig. 5B) showed simila EGFP exp ession, indica ing ha he ans-
ec ion e iciency o ha o mula ion main ained s able o e a mon h.
Addi ionally, he ans ec ion e iciency o esh and 30 days s o ed
nanodiasomes was also e alua ed in ano he CNS cell ype, speci ically
in a p ima y neu onal cell cul u e, which clea ly co obo a ed he high
gene deli e y capaci y, by means o EGFP exp ession, o s o ed o mu-
la ions o e a mon h and e en 3 mon hs (Supplemen a y Fig. S1).
Fig. 3. Gene deli e y e iciency and oxici y o o mula ions in HEK-293 cells 48 h a e ans ec ion wi h nanodiaplexes and nioplexes a 5/1 ca ionic lipid/DNA
a io (w/w) o e ime a 4˚C and 25˚C. A. No malized pe cen ages o EGFP posi i e li e cells (ba s) and cell iabili y (do s) ob ained by low cy ome y. B. Mean
luo escence in ensi y alues ob ained by low cy ome y. Each alue ep esen s he mean ±SD o 3 measu emen s. C-D. Me ged images showing EGFP signal in
HEK-293 ans ec ed cells wi h bo h complexes a 5/1 lipid/DNA a io (w/w) a day 0 (C) and a e 30 days o s o age a 4
◦C (D). Scale ba s: 200
μ
m. *** P <0.001;
** P <0.01 o nanodiaplexes s nioplexes, no nega i e signi ican di e ences in e m o li e cells (%) o nioplexes be ween day 0 and he es o days and
empe a u es; # P <0.05 o nioplexes be ween day 0 and he es o days and empe a u es; $ P <0.05 o nanodiaplexes a day 30 compa ed wi h he es o days
and empe a u es.
Fig. 4. Cellula up ake in HEK-293 cells 4 h a e exposu e o nanodiasomes and niosomes a 5/1 lipid/DNA a io (w/w) a day 0 and a e 30 days o o mula ions
s o age a 4˚C. A. No malized pe cen ages o FITC-pEGFP posi i e li e cells a e he exposu e o hese complexes. Each alue ep esen s he mean ±SD o 3
measu emen s. B. Con ocal mic oscopy images. Cell nuclei we e colo ed in blue (DAPI); F-ac in in ed (Phalloidin); nanodiaplexes and nioplexes in g een (FITC).
Scale ba s: 50
μ
m.* P <0.05 o nanodiaplexes s nioplexes. (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.)
N.H. AL Q aish e al.
In e na ional Jou nal o Pha maceu ics 639 (2023) 122968
6
3.5. In i o ans ec ion e iciency o nanodiaplexes
Nanodiaplexes, p epa ed wi h esh and s o ed nanodiasomes, we e
adminis e ed o he mouse eye h ough in a i eal (Fig. 6A and C) and
sub e inal injec ions (Fig. 6B and D), and luo escence signal was
de ec ed in di e en e inal cell laye s a e one week. Bo h sub e inally
and in a i eally adminis e ed nanodiaplexes showed ha EGFP
exp ession colocalized mainly wi h mic oglial ma ke Iba-1 and was also
loca ed in he ganglion cell laye (GCL), as well as in he inne nuclea
laye (INL) wi h some di used luo escence signal in he ou e nuclea
laye (ONL), and e en he e inal pigmen epi helial cell laye (RPE)
a e sub e inal injec ions (Fig. 6B and D). Resul s also showed ha he
in ensi y o he luo escence signal was compa able in bo h ans ec ions
wi h eshly p epa ed and 30 days s o ed o mula ions. Addi ionally,
mouse e inal cells ole a ed well he exposi ion o nanodiaplexes, in
e ms o cell iabili y, conside ing he esul s epo ed in he quali a i e
analysis.
4. Discussion
The high e sa ili y o non- i al ec o s elies on he la ge a ie y o
a ailable nanoma e ials and p epa a ion me hods ha can be employed.
Among he wide ple ho a, NDs ha e been ecognized as powe ul ools
o inc ease he ans ec ion e iciency o many non- i al ec o sys ems
due o hei unique physicochemical p ope ies, including e sa ile
su ace chemis y and ease o unc ionaliza ion, oge he wi h hei high
biocompa ibili y and low oxici y (Al Q aish e al., 2022). In addi ion,
NDs show a a ou able pa icle dis ibu ion, being almos sphe ical in
shape. In e es ingly, hey can also be easily unc ionalized wi h many
chemical compounds, show a high su ace a ea- o- olume a io, and
hei p oduc ion p ocess can be easily scalable (K üge e al., 2006; Liu
e al., 2007). As NDs p esen low s abili y in suspension, hei combi-
na ion wi h niosome o mula ions could be necessa y o p o ide
enhanced s abili y, which is necessa y o gene deli e y applica ions.
The e o e, in his wo k we combined NDs wi h a niosome o mula ion,
based on a ca ionic lipid and non-ionic su ac an , ob aining a inal
o mula ion named nanodiasome in o de o assess o e ime a se e al
s o age empe a u es he s abili y o nanodiasomes compa ed wi h
niosomes de oid o NDs. To e alua e he s abili y o he o mula ions,
ele an physicochemical pa ame e s ha a ec o he ans ec ion
p ocess, along wi h biocompa ibili y and ans ec ion e iciency s udies
we e pe o med in in i o and in i o condi ions.
The physicochemical cha ac e is ics cons i u e key pa ame e s ha
de e mine he biological beha iou o he o mula ions, including hei
cellula in e naliza ion p ocess, gene deli e y e iciency and biocom-
pa ibili y. In he p esen wo k, nanodiasomes showed a sligh ly highe
mean pa icle size han niosomes a day 0, p obably due o he inco -
po a ion o NDs as addi ional elemen s, which migh ha e a ec ed o he
packing o he o mula ion. The lowe dispe si y alues obse ed wi h
nanodiasomes, indica ed a mo e homogeneous pa icle size dis ibu ion
o ha o mula ion compa ed o he niosome o mula ion. Bo h o -
mula ions p esen ed s a is ically ele an oscilla ions in hei physico-
chemical pa ame e s, especially a e 30 days being s o ed a 25 ◦C,
sugges ing ha hese pa ame e s a e be e p ese ed i o mula ions a e
kep a 4 ◦C a he han a highe empe a u es. Hence, in gene al e ms,
i can be said ha nanodiasomes a e physicochemically mo e s able o e
ime han niosomes. The e o e, NDs in eg a ion in he lipid s uc u e o
niosomes is in ol ed in supplying highe s abili y o he o mula ion,
p obably p o iding mo e igidi y, by a ec ing he a angemen o he
lipid memb ane and modi ying he heological and packing beha iou o
he o mula ion (Sainz-Ramos e al., 2021).
A e he e alua ion o physicochemical p ope ies, biological in i o
ans ec ion s udies we e pe o med in HEK-293 cells. We ound ha
ans ec ed cells wi h nanodiaplexes p esen ed highe cell iabili y
alues han he ones ans ec ed wi h nioplexes, which sugges s ha he
o mula ions based on nanodiasomes a e be e ole a ed by hese cells.
These esul s a e in acco dance wi h he p e iously epo ed high
biocompa ibili y and low oxici y o NDs (K üge e al., 2006; Liu e al.,
2007; Zhang e al., 2009). In addi ion, he gene deli e y e iciency o
nanodiaplexes was app oxima ely 4- old supe io han he one o nio-
plexes, and his di e ence was main ained o e ime. The MFI alues
e e o he quan i y o he exp essed GFP p o ein, and also indica ed a
highe ans ec ion capaci y o nanodiaplexes o e nioplexes. In his
sense, i is no ewo hy ha he numbe o DNA copies pe cell dec eased
p og essi ely o nioplexes om day 15, while nanodiaplexes did no
su e any al e a ion in his pa ame e un il day 30. Taken all oge he ,
his could sugges ha he combina ion o NDs wi h niosomes enhances
he s abili y o he o mula ion, achie ing mo e consis en and suc-
cess ul ans ec ion esul s o e ime. To be e unde s and he di e -
ences obse ed be ween bo h o mula ions, we s udied hei cellula
up ake a 0 and 30 days a e being s o ed a 4 ◦C. We ound s a is ically
ele an di e ences in he pe cen age o cellula up ake be ween bo h
Fig. 5. EGFP signal in p ima y cul u e o a e inal ans ec ed cells wi h eshly p epa ed (A) and 30 days s o ed a 4
◦C (B) nanodiasomes a 5/1 lipid/DNA a io
(w/w). Scale ba : 40 µm. Blue: Hoechs 33,342 (cell nuclei); G een: EGFP. Scale ba s: 40
μ
m. (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.)
N.H. AL Q aish e al.
In e na ional Jou nal o Pha maceu ics 639 (2023) 122968
7
o mula ions, ob aining almos 100% no malized alues o cellula up-
ake wi h nanodiasomes and a ound 70% wi h niosomes a bo h s o age
condi ions, which could suppo he idea ha NDs inc ease he igidi y
o he niosome o mula ion, enhancing he cellula en y (Manzana es
and Cena. 2020). This highe cellula up ake could in pa explain he
di e ences in ans ec ion e iciency be ween he wo o mula ions, bu
u he aspec s need also o be aken in o accoun . T adi ionally, cellula
endocy osis o non- i al ec o s is media ed h ough he endosomal
pa hway, which e en ually leads o endosomal esicles wi h an acidic
en i onmen and diges i e enzymes (Agi e e al., 2015). In hese esi-
cles, he DNA isks o being deg aded be o e eaching he nucleus.
The e o e, DNA endosomal escape becomes a key s ep in o de o ach-
ie e success ul gene deli e y. In his sense, i has been epo ed ha NDs
a e able o escape he endosome con inemen by up u ing he esicle
memb ane sho ly a e hei cellula up ake (Chu e al., 2015), which
would also con ibu e o jus i y he highe gene deli e y e iciency o
nanodiaplexes compa ed o nioplexes coun e pa s. In addi ion, u he
ans ec ion assays in CNS cells, bo h e inal and neu onal p ima y cells,
con i med e ec i e ansgene exp ession a e ans ec ion wi h bo h
eshly p epa ed and 30 days s o ed nanodiaplexes.
The e o e, based on hese esul s, we pe o med an in i o assay in
o de o de e mine he gene deli e y e iciency o nanodiaplexes om
esh and 30 days s o ed nanodiasomes o mula ion in mouse e ina.
Fo mula ions we e injec ed h ough in a i eal and sub e inal ou es,
which a e widely used in o he clinic o he ea men o gene ically
based e inal diso de s (Conley and Naash. 2010). In mos cases, a e
in a i eal injec ion, ganglion cell laye o he e ina shows high
ansgene exp ession le els (Fa jo e al., 2006), which, o ins ance,
could be in e es ing o he ea men o glaucoma, a highly p e alen
inhe i ed e inal diso de ha causes blindness (Almasieh e al., 2012;
Kachi e al., 2005). On he o he hand, he mo e in asi e sub e inal
ou e is use ul o ans ec ing he ou e laye o he e ina (Almasieh
e al., 2012; Kachi e al., 2005), which would be in e es ing o ace
e inal diseases ela ed o mu a ions a he pho o ecep o s and he
e inal pigmen epi helium le el, such as Lebe ’s congeni al amau osis,
S aga d ’s disease o e ini is pigmen osa (Lipinski e al., 2013). In he
p esen s udy, EGFP signal was ound mainly in mic oglial cells. Such
exp ession was also loca ed in bo h, he inne and ou e laye s o he
e ina bo h a e in a i eal and sub e inal injec ion o nanodiaplexes,
which sugges ha his o mula ion is able o e icien ly di use along he
di e en e inal laye s achie ing high ansgene exp ession a di e en
le els, which would be ele an om he he apeu ic poin o iew. In
Fig. 6. In i o assays showing EGFP signal in mouse e ina a e in a i eal (IV) (A-C) and sub e inal (SR) (B-D) adminis a ion o eshly p epa ed (A-B) and 30 days
s o ed (C-D) nanodiasomes ec o ing EGFP plasmid a 5/1 lipid/DNA a io (w/w). Blue: Hoechs 33,342 (cell nuclei); G een: EGFP; Red: Iba-1. OS: ou e segmen s;
ONL: ou e nuclea laye ; INL; inne nuclea laye ; GCL: ganglion cell laye . Scale ba : 20
μ
m. (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.)
N.H. AL Q aish e al.
In e na ional Jou nal o Pha maceu ics 639 (2023) 122968
8
addi ion, esul s e ealed high EGFP exp ession in i o a e he
adminis a ion o 30 days s o ed o mula ion, indica ing ha he s o age
o he o mula ion a 4 ◦C o 30 days does no a ec i s ans ec ion
e iciency.
5. Conclusions
Taken oge he , he main conclusions o he p esen wo k a e ha (i)
nanodiasomes p esen highe mean pa icle size, lowe dispe si y and
highe ze a po en ial alues han niosomes, (ii) nanodiasomes p ese e
mo e cons an hei physicochemical pa ame e s o e ime han nio-
somes and bo h o mula ions p e e low empe a u es o s o age, (iii)
nanodiaplexes p esen an a ound 4- old supe io ans ec ion e iciency
han nioplexes, in e ms o pe cen age o li e ans ec ed cells, al hough
bo h main ain hei ans ec ion e iciency o e ime, (i ) nanodiaplexes
a e mo e e icien ly up aken by HEK-293 cells han nioplexes, ( ) high
gene deli e y e iciency o nanodiaplexes is main ained o e ime in a
cen al ne ous p ima y cell cul u es and ( i) also in i o a e sub e inal
and in a i eal injec ion o nanodiaplexes in mouse e ina.
CRediT au ho ship con ibu ion s a emen
Nuseibah H. AL Q aish: In es iga ion, Me hodology, Visualiza ion,
W i ing – o iginal d a . Ilia Villa e-Bei ia: Fo mal analysis, Visuali-
za ion, W i ing – o iginal d a . Idoia Gallego: Fo mal analysis, Visu-
aliza ion, W i ing – e iew & edi ing. Gema Ma ínez-Na a e e:
In es iga ion, Visualiza ion, W i ing – e iew & edi ing. C is ina So o-
S´
anchez: In es iga ion, Visualiza ion, W i ing – e iew & edi ing.
My iam Sainz-Ramos: In es iga ion, W i ing – e iew & edi ing. Tania
B Lopez-Mendez: . Alejand o J. Pa edes: . F ancisco Ja ie Chich´
on:
Fo mal analysis, Da a cu a ion. Noelia Zama e˜
no: Me hodology.
Edua do Fe n´
andez: Supe ision, W i ing – e iew & edi ing. Gus a o
Pu as: Concep ualiza ion, Supe ision, P ojec adminis a ion, W i ing
– e iew & edi ing. Jos´
e Luis Ped az: Concep ualiza ion, Supe ision,
P ojec adminis a ion, W i ing – e iew & edi ing, Funding acquisi ion.
Decla a ion o Compe ing In e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Da a a ailabili y
The da a ha has been used is con iden ial.
Acknowledgemen s
This wo k was suppo ed by he Basque Coun y Go e nmen
(Consolida ed G oups, IT1448-22 and by CIBER -Conso cio Cen o de
In es igaci´
on Biom´
edica en Red- CB06/01/1028, Ins i u o de Salud
Ca los III, Minis e io de Ciencia e Inno aci´
on. Au ho s wish o hank:
ICTS “NANBIOSIS”, speci ically he D ug Fo mula ion Uni (U10) o he
CIBER in Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN)
o he in ellec ual and echnical assis ance. Au ho s also hank SGIke
(UPV/EHU) o echnical and human suppo . Au ho s acknowledge
Rocio A anz, access o he c yoEM CSIC acili y in he con ex o he
CRIOMECORR p ojec (ESFRI-2019-01-CSIC-16). I.V.B. hanks he
Uni e si y o he Basque Coun y (UPV/EHU) o he g an ed pos -
doc o al ellowship (call o he Specializa ion o Doc o Resea che
Pe sonnel o he UPV/EHU, g an e e ence: ESPDOC19/47). M.S.R.
hanks he Uni e si y o he Basque Coun y (UPV/EHU) o he g an ed
p e-doc o al ellowship (PIF17/79).
Appendix A. Supplemen a y ma e ial
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.ijpha m.2023.122968.
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