Ionizable nanoemulsions for RNA delivery into the central nervous system – importance of diffusivity

Jou nal o Con olled Release 372 (2024) 295–303
A ailable online 24 June 2024
0168-3659/© 2024 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC license (h p://c ea i ecommons.o g/licenses/by-
nc/4.0/).
Ionizable nanoemulsions o RNA deli e y in o he cen al ne ous sys em –
impo ance o di usi i y
Mi eya L. Bo ajo
a
,
b
,
c
, Aloia Quijano
a
,
c
, Philipp Lapuhs
a
,
b
,
c
, Ana I. Rod iguez-Pe ez
a
,
c
,
d
,
Shubaash An hiya
a
,
b
,
c
, Jos´
e L. Labandei a-Ga cia
a
,
c
,
d
, Ri a Valenzuela
a
,
c
,
d
,
*
, Ma ía
Jos´
e Alonso
a
,
b
,
c
,
*
a
Cen e o Resea ch in Molecula Medicine and Ch onic Diseases (CiMUS), A . Ba celona s/n, Campus Vida, Uni e si y de San iago de Compos ela, 15782 San iago de
Compos ela, Spain
b
Depa men o Pha macy and Pha maceu ical Technology, School o Pha macy, Uni e si y o San iago de Compos ela, 15782 San iago de Compos ela, Spain
c
IDIS Resea ch Ins i u e, Uni e si y o San iago de Compos ela, 15782 San iago de Compos ela, Spain
d
Ne wo king Resea ch Cen e o Neu odegene a i e Diseases (CIBERNED), Mad id, Spain
ARTICLE INFO
Keywo ds:
B ain deli e y
Di usi i y
mRNA
Nanoemulsion
RNA he apeu ics
siRNA
ABSTRACT
Lipid nanopa icles (LNPs) cu en ly domina e he RNA deli e y landscape; howe e hei limi ed di usi i y
hampe s a ge ed issue dissemina ion, and, hence, hei capaci y o in acellula d ug deli e y. This is espe-
cially ele an o issues such as he cen al ne ous sys em (CNS), whe e o e coming p oac i e b ain ba ie s is
c ucial o he e icacy o gene ic he apeu ics. This esea ch aimed o c ea e ionizable nanoemulsions (iNEs), a
new gene a ion o RNA deli e y sys ems wi h enhanced di usi i y. The de eloped iNEs (consis ing o he
combina ion o C12–200, DOPE, Vi amin E, and DMG-PEG) wi h a size below 100 nm, neu al su ace cha ge,
and high RNA loading capaci y, showed excellen cell iabili y and ans ec ion e iciency in a ious cellula
models, including neu ons, as ocy es, and mic oglia. Subsequen ly, iNEs con aining mRNA GFP we e es ed o
CNS ans ec ion, highligh ing hei excep ional di usi i y and selec i e ans ec ion o neu ons ollowing in a-
pa enchymal adminis a ion.
1. In oduc ion
Cu en ly, he po en ial o RNA he apeu ics o ea ing mul iple
medical condi ions hea ily elies on nano echnology. Among nano-
ca ie s, lipid nanopa icles (LNPs) a e he sole ma ke -app o ed de-
li e y sys em o messenge RNA (mRNA) and small in e e ing RNA
(siRNA) deli e y [1]. The i s LNP-based gene ic he apeu ic, Onpa -
o® (Alnylam), was app o ed in 2018 o he ea men o he edi a y
ans hy e in-media ed amyloidosis [2]. In la e 2020, mRNA accines,
Comi na y® (BioNTech/P ize ) and Spike ax® (Mode na), we e
app o ed agains he se e e acu e espi a o y synd ome co ona i us 2
(SARS-CoV-2), he cause o he co ona i us disease 2019 (COVID-19)
pandemic [3].
In hese o mula ions, LNPs encapsula e mRNA wi h componen s
including an ionizable lipid, choles e ol, a helpe lipid, and a PEGyla ed
lipid [4]. Ionizable lipids a e esponsible o he complexa ion o he
RNA. They possess he abili y o ansi ion om neu al o posi i ely
cha ged in acidic endosomal pH, p omo ing usion wi h he endosomal
memb ane and ca go elease in he cy oplasm o he a ge ed cells [5–7].
While he unsa u a ed ionizable lipid DLin-MC3-DMA was used in
Onpa o®, u he op imiza ion in oduced mul i- ail ionizable lipids in
he esea ch ield, such as C12–200, enhancing endosome dis up ion
[8–10]. Un o una ely, he deg adabili y o hese ionizable lipids e-
mains a limi a ion, mainly due o hei s able backbones [11]. Recen
ad ances in he ield ocus hei e o s on he de elopmen o he nex
gene a ion o ionizable lipids, aiming o enhance hei ans ec ion
pe o mance while imp o ing hei biodeg adabili y, ensu ing hei
o e all sa e y and low oxici y p o ile [12–14].
When designing a nanoca ie , success ul in i o ans ec ion is no
he sole equi emen o nanoca ie design. A e adminis a ion, he
nanoca ie mus each he in ended issue, di use ac oss i , and e ec-
i ely each he a ge ed cell. The limi ed di usion o LNPs in he cen al
* Co esponding au ho s a : Cen e o Resea ch in Molecula Medicine and Ch onic Diseases (CiMUS), A . Ba celona s/n, Campus Vida, Uni e si y de San iago de
Compos ela, 15782 San iago de Compos ela, Spain.
E-mail add esses: [email p o ec ed] (R. Valenzuela), [email p o ec ed] (M.J. Alonso).
Con en s lis s a ailable a ScienceDi ec
Jou nal o Con olled Release
jou nal homepage: www.else ie .com/loca e/jcon el
h ps://doi.o g/10.1016/j.jcon el.2024.06.051
Recei ed 14 May 2024; Accep ed 20 June 2024
Jou nal o Con olled Release 372 (2024) 295–303
296
ne ous sys em (CNS) has been ex ensi ely no ed. Fo example, s udies
on LNP di usion in he b ain e ealed educed PTEN (phospha ase and
ensin homolog-1) le els in neu ons wi hin 1 mm a e he adminis a-
ion o LNPs con aining PTEN-siRNA [15]. This was co obo a ed by
Cy5-labeled mRNA LNPs adminis e ed in he s ia um, de ec ing signals
1–1.5 mm om he injec ion si e [16]. Un o una ely, he majo i y o
s udies quan i ying nucleic acids in he b ain do no p o ide in o ma ion
abou hei speci ic localiza ion ac oss he issue [17,18].
The hypo hesis o his wo k was ha he limi ed di usi i y o LNPs
ac oss he b ain may s em om hei dense co e and ela i e igidi y
[19]. The same may apply o o he solid-co e nanopa icles [20,21].
Hence, ou objec i e was cen e ed on he de elopmen o so e , luid,
and de o mable co es, such as in nanoemulsions (NEs). NEs, wi h
nanome ic d ople sizes, ypically consis o an oil-in-wa e suspension
o oils and su ac an s [22,23]. Up un il now, NEs ha e been ex ensi ely
used o he solubiliza ion o small hyd ophobic molecules and some
p elimina y e o s ha e been o ien ed o accommoda e RNA molecules
[24]. Fo example, some au ho s ha e ound ha inco po a ing ca ionic
lipids and su ac an s in o NEs allowed RNA abso p ion, and p oposed
hei use in he ield o mRNA accina ion [25–27]. In addi ion, ca ionic
NEs con aining 1,2-dioleoyl-3- ime hylammonium p opane (DOTAP)
we e epo ed o he in anasal adminis a ion o siRNA an i- umo
nec osis ac o -
α
(TNF-
α
), showing a posi i e educ ion o an i-
in lamma o y ma ke s in he b ain. [28]. Simila ly, a DOTAP-
con aining NE, in anasally deli e ed siRNA CD73 o glioblas oma
ea men , signi ican ly educed umo g ow h in an animal model [29].
Howe e , none o hese s udies elucida ed he ole o de o mabili y o
pa icle composi ion o he posi i e esul s on success ul d ug deli e y
ac oss he b ain. Aligned wi h he so and luid co e concep , s udies
indica e ha bo h he pa icle size and in e nal composi ion o he
nanoca ie s can imp o e di usion p ope ies. Fo example, di usi i y
s udies in mucus and b ain demons a ed ha PEGyla ion o nano-
pa icles can enhance hei di usi i y p o ile in a size-dependen
manne [30–32].
He e, we le e age he knowledge o ou lab in NEs and nanocapsules
(NCs) deli e y pla o ms o de elop he nex gene a ion o ionizable NEs
(iNEs) [33–39]. We explo ed a ious PEGyla ed lipid leng hs o op imal
ans ec ion e iciency and low oxici y in i o. Addi ionally, cy o ox-
ici y p o iles and ans ec ion e iciency we e assessed in neu onal,
mic oglial, and as ocy ic cellula models. To alida e ou hypo hesis on
enhanced di usi i y o iNEs, in a-pa enchymal adminis a ion in a s
using iNEs encapsula ing GFP mRNA was pe o med. This aimed o
elucida e speci ic CNS cells exp essing GFP and he di usi i y p o ile o
iNEs, o hei possible applica ion as gene deli e y sys ems o he
ea men o a ious CNS diseases.
2. Ma e ials and me hods
2.1. Ma e ials
C12–200, HCl sal (1, 1
′
-((2-(4-(2-((2-(bis(2-hyd oxydodecyl)amino)
e hyl)(2-hyd oxydodecyl)amino)e hyl)pipe azin-1-yl)e hyl)azanediyl)
bis(dodecan-2-ol)) and DMG-PEG
2000
((R)-me hoxy-polye hyleneglycol-
2000-ca bamoyl-di-O-my is yl-sn-glyce ide) we e kindly gi ed by
Mu hiah Manoha an, om Alnylam Pha maceu icals (MA, USA). DOPE
(1,2-dioleoyl-sn-glyce ol-3-phosphoe hanolamine) was pu chased om
A an i Pola Lipids (AL, USA). Vi amin E (Vi E) (D, L-
α
- ocophe ol) was
ob ained om BASF (Mannheim, Ge many). siGFP (siRNA an i-GFP)
was acqui ed om BioSp ing GmbH (F ank u , Ge many). mGFP
(mRNA encoding o GFP, CleanCap EGFP mRNA) was pu chased om
T ikLink Bio echnologies (CA, USA).
2.2. Fo mula ion o iNEs
iNEs we e p epa ed in a single s ep, using a mic o luidic mixe
NanoAssembl ™ bench- op ins umen , P ecision NanoSys ems Inc.
(Vancou e , Canada), ollowing a sol en displacemen echnique [40].
Fo mula ion o he iNE and complexa ion o he oligonucleo ide ca go
occu ed simul aneously. In summa y, 0.2 mL o he o ganic phase
(12.8 mg/mL o C12–200, 3.7 mg/mL o DOPE, 6 mg/mL o Vi amin E,
and 2 mg/mL o DMG-PEG
2000
in E OH, leading o mola a io o 35:16:
46.5: 2.5) we e mixed wi h 1 mL o he aqueous phase (con aining 0.24
mg/mL o he selec ed RNA dilu ed in ci a e bu e , pH 4, 10 mM).
The ni ogen- o-phospha e a io (be ween he amine g oups o
C12–200 and he phospha e g oups o he RNA) was main ained a 15:1.
The Flow Ra e Ra io was 1:5 (o ganic o aqueous phase). The To al Flow
Ra e was main ained a 12 mL/min. The esul ing iNE-RNA o mula ions
p esen ed a heo e ical RNA concen a ion o 0.2 mg/mL. Samples we e
se aside o s abiliza ion o 10 min be o e physicochemical
cha ac e iza ion.
iNEs con aining Tween 80® we e p epa ed by bulk mixing, in a
single s ep. B ie ly, 0.2 mL o he o ganic phase (12.8 mg/mL o
C12–200, 3.7 mg/mL o DOPE, 6 mg/mL o Vi amin E, and 1 mg/mL o
Tween 80® in E OH, leading o mola a io o 35: 16: 46.5: 2.5) we e
added o e 1 mL o he aqueous phase (con aining 0.24 mg/mL o RNA
dilu ed in ci a e bu e 10 mM, pH 4). The aqueous phase was unde
s i ing a 1400 pm, and he esul ing solu ion was kep unde agi a ion
o 5 s. Then, i was le o s abilize o 10 min be o e physicochemical
cha ac e iza ion.
2.3. Physicochemical cha ac e iza ion o iNEs
The hyd odynamic diame e and polydispe se index (PDI) we e
cha ac e ized by dynamic ligh sca e ing (Ze asize ® Nano ZS, Mal e n
Ins umen s, Mal e n, UK). ζ-po en ial was measu ed in e ms o mean
elec opho e ic mobili y alues, measu ed by lase Dopple elec o-
pho esis wi h he same equipmen . Pa icle size and PDI measu emen s
we e pe o med a e dilu ing he samples 10- old in 1×PBS, pH 7.
ζ-po en ial cha ac e iza ion was pe o med a e dilu ion o samples 20-
old in RNase- ee wa e .
Encapsula ion e iciency (EE%) was de e mined ollowing di e en
me hodologies. In all cases, iNE-RNA o mula ions we e dilu ed in a 1:1
( / ) a io wi h T i on X-100 o hepa in solu ions, p epa ed a 50 mg/
mL in RNase- ee wa e . Incuba ion wi h T i on X-100 (Sigma-Ald ich,
MO, USA) induced he dis up ion o he nanoca ie , while hepa in
(Sigma-Ald ich, MO, USA) led o he displacemen o he RNA om he
nanopa icle. Aga ose gel elec opho esis was used o quali a i ely
assess he amoun o RNA encapsula ed in iNE-RNA o mula ions.
Samples con aining 1–3
μ
g o RNA we e loaded in an aga ose gel a 1%
w/ in T is Ace a e-EDTA bu e (Sigma-Ald ich, MO, USA) be o e and
a e incuba ion wi h T i on X-100 and hepa in. Samples we e dilu ed
wi h equal olumes o loading mix, con aining 6×SYBR® Gold nucleic
acid s ain (In i ogen, CA, USA), 44% Glyce ol, and 0.176 g/L B o-
mophenol blue. F ee RNA was included as a con ol. Gels we e un o
30 min a 90 V in Sub-Cell GT cell 96/192 (Bio-Rad Labo a o ies, CA,
USA), and e alua ed wi h a UV ansillumina o imaging sys em (Mo-
lecula Image ® Gel Doc™ XR, Bio-Rad Labo a o ies, CA, USA).
To quan i a i ely de e mine he EE% o iNE-RNA o mula ions,
Quan -iT RiboG een RNA assay ki (In i ogen, MA, USA) was used.
B ie ly, samples we e dilu ed 50- old in TE bu e (1×), ollowed by a 2-
old dilu ion wi h T i on X-100 and incuba ion a 37 ◦C o 30 min.
Subsequen ly, samples we e dilu ed 10- old wi h TE bu e (1×). The
s anda d cu e was p epa ed using RNA a concen a ions be ween 0.1
and 1
μ
g/mL, in he p esence o T i on X-100. Following manu ac u e
ins uc ions, RiboG een eagen was dilu ed 200- old in TE bu e (1×),
and added o an equal olume o sample, leading o a inal olume o
200
μ
L. Samples and he s anda d cu e we e ans e ed o a 96-black
polys y ene eade (Syne gy H1, BioTek Ins umen s, VT, USA), using
exci a ion a 485 nm and emission a 530 nm. The s anda d cu e (
2
≥
0.99) was used o de e mining he RNA concen a ion o he sample.
The encapsula ion e iciency o RNA was calcula ed acco ding o he
ollowing Eq. 1.
M.L. Bo ajo e al.
Jou nal o Con olled Release 372 (2024) 295–303
297
Encapsula ion e iciency (EE%) =
F ee o
unencapsula ed RNA
To al RNA ×100
Whe e [RNA]dis up ed is he RNA concen a ion de e mined a e
ea ing he nanosys ems wi h he dis up ing agen . [RNA] heo e ical is he
heo e ical o al RNA concen a ion o he nanosys em.
Equa ion 1 Calcula ion o encapsula ion e iciency (EE%).
2.4. In i o iNE-siGFP ans ec ion e iciency and cy o oxici y in HeLa
cells
Assessmen o cy o oxici y and ans ec ion e iciency was pe o med
in HeLa cells exp essing GFP, which we e kindly gi ed by P o . Ja ie
Mon eneg o (Cen e o Resea ch in Biological Chemis y and Molecula
Ma e ials, CiQUS, San iago de Compos ela, Spain). A o al o 10,000
cells we e seeded pe well in a la bo om 96-well pla e and allowed o
adhe e o 24 h. Cells we e ea ed wi h iNE-siGFP o 4 h, in Op i-
MEM™ (Gibco™, The mo Fishe , MA, USA) a siGFP concen a ions
anging om 250 o 10 nM pe well. Nanoca ie s we e hen emo ed
and eplaced wi h a comple e medium (DMEM supplemen ed wi h 10%
e al bo ine se um). Cells we e incuba ed o ano he 20 h. Cell iabili y
was measu ed by luo escence-based esazu in assay [41]. B ie ly, cells
we e incuba ed wi h esazu in eagen (Resazu in sodium sal , Sigma-
Ald ich, MO, USA) supplemen ed comple e media o 45 min, and he
esul ing luo escence was measu ed in a pla e eade a 544/590 nm.
Cells we e hen ypsinized, ha es ed, and ixed wi h 1% (w/ ) o m-
aldehyde in PBS. The pe cen age o GFP-nega i e cells and mean luo-
escence in ensi y (MFI) we e analyzed by low cy ome e y (BD Accu i,
BD Biosciences, NJ, USA).
2.5. In i o iNE-mGFP ans ec ion e iciency and cy o oxici y in neu on,
as ocy e, and mic oglia models
In i o cy o oxici y and ans ec ion e iciency we e e alua ed in
human neu onal (SH-SY5Y cell line), a as ocy ic (C6 glioma cell line),
and human mic oglial (CHME-3 cell line) models. Fo ha , a o al o
150,000 cells we e seeded pe well in a la bo om 12-well pla e and
allowed o adhe e o 24 h. Cells we e ea ed wi h iNE-mGFP in Op i-
MEM™ (Gibco™, The mo Fishe , MA, USA) a a concen a ion o 5
μ
g
pe well, o 4 h. Nanoca ie s we e hen emo ed and eplaced wi h a
comple e medium. Cells we e incuba ed o ano he 20 h. Cell iabili y
was measu ed by abso bance-based MTT assay (3-(4, 5-dime hyl-2- hia-
zolyl)-2, 5-diphenyl-2H- e azolium b omide; Sigma-Ald ich, MO, USA).
B ie ly, cells we e incuba ed wi h MTT eagen (1 mg/mL) o 4 h a
37 ◦C. A e emo ing he MTT eagen , he esul ing o mazan c ys als
we e dissol ed in acidic isop opanol and quan i ied in a pla e eade a
544/590 nm (In ini e 200 PRO, Tecan, M¨
annedo , Swi ze land). Fo
ans ec ion e iciency assessmen , cells we e ypsinized, ha es ed,
and ixed wi h 1% (w/ ) o maldehyde in PBS. The pe cen age o GFP-
posi i e cells and mean luo escence in ensi y we e analyzed by low
cy ome y (BD Accu i, BD Biosciences, NJ, USA).
2.6. Animal s udies
The animal s udy p o ocol was ca ied ou ollowing he Eu opean
Communi ies Council Di ec i e 2010/63/EU, Di ec i e 86/609/EEC,
and Spanish RD 526/2014, and was app o ed by he co esponding
commi ee a he Uni e si y o San iago de Compos ela (p o ocol
14,715,012/2021/012; las e sion 16 Ap il 2021).
2.7. Assessmen o di usi i y p o ile o iNE-mGFP upon in a-
pa enchymal adminis a ion
Adminis a ion o iNEs was pe o med in male Sp age-Dawley a s (3
animals in he iNE-mGFP g oup and 1 animal in he PBS, con ol g oup)
deeply anes he ized wi h ke amine (50 mg/kg) and mede omidine (0.4
mg/kg). Anes he ized animals we e moun ed in a s e eo axic ame
(Kop Ins umen s, CA, USA) and we e injec ed wi h 3
μ
g o mGFP
encapsula ed on o iNEs, in a o al olume o 3
μ
L. The solu ion was
injec ed using a 5-
μ
L Hamil on sy inge, coupled o a mo o ized injec ion
(S oel ing), a a a e o 0.5
μ
L/min. S e eo axic coo dina es we e A/P:
0.8 mm, M/L: 3.0 mm and D/V: 5.0 mm. The needle used o he
adminis a ion was le in place o an addi ional 5 min be o e wi h-
d awal o a oid iNEs e lux. A e 24 h, animals ecei ed an anes he ic
o e dose and hey we e sac i iced by decapi a ion; hei b ains we e
apidly emo ed, c yop o ec ed, and cu in o co onal issue sec ions
using a sliding mic o ome. Sec ions we e p ocessed o immuno luo-
escence labeling as ollows.
F ee- loa ing issue sec ions we e p e-incuba ed in KPBS-1% BSA
wi h 5% no mal donkey se um (Sigma-Ald ich, MO, USA) and 0.03%
T i on X-100 o 60 min a oom empe a u e. Ini ially, single immu-
no luo escence was pe o med o iden i y cells capable o exp essing
GFP a e mGFP ans ec ion. The di e en b ain cell ypes we e labeled
wi h p ima y an ibodies agains class III β- ubulin (β III- ubulin, 1:750,
T8660, Sigma-Ald ich, MO, USA) as a neu onal make , o glial ib illa y
acidic p o ein (GFAP, 1:500, MAB360, Me k Millipo e, MA, USA) as an
as ocy ic ma ke , o ionized calcium-binding adap o molecule 1 (Iba-
1, 1:500, 019–19,741, Wako Chemicals, Neuss, Ge many) as a mic oglial
make . Cell nuclei we e ma ked wi h he DNA-binding dye Hoechs
33342 (1:2000, 62,249, Sigma-Ald ich, MO, USA). Immuno eac ion was
isualized wi h he luo escence seconda y an ibody Alexa Fluo 568-
conjuga ed donkey an i- abbi IgG (1:200, Molecula P obes, OR,
USA) o Alexa Fluo 488-conjuga ed donkey an i-mouse IgG (1:200,
Molecula P obes, OR, USA). Sec ions we e moun ed on gela in-coa ed
slides and co e -slipped wi h Immumoun (The mo-Shandon). Co-
localiza ion o hese ma ke s was assessed by con ocal lase mic o-
scopy (AOBS-SP5X; Leica Mic osys ems Heidelbe g GmbH, Mannheim,
Ge many).
Then, conside ing he au o-g een luo escence depic ed by neu ons
[42], he decision was made o pe o m a double immuno luo escence
agains GFP (an i-GFP, 1:1000, C10362, Molecula P obes, OR, USA)
and β III- ubulin, ollowed by he incuba ion wi h luo escence sec-
onda y an ibodies Alexa Fluo 568-conjuga ed donkey an i- abbi IgG
(1:200, Molecula P obes, OR, USA) and Alexa Fluo 488-conjuga ed
donkey an i-mouse IgG (1:200, Molecula P obes, OR, USA), espec-
i ely. Then, he p epa a ion o he sec ion was pe o med as p e iously
desc ibed.
3. Resul s and discussion
To maximize gene he apy e ec i eness, nanoca ie s mus no only
sa egua d RNA ca go bu also enable e icien di usion ac oss a ge ed
issues, especially in challenging issues such as he CNS. Fo he design
o nanosys ems wi h enhanced di usi i y, we explo ed combining
lipids, which a e ypical componen s o LNPs wi h oily co es om NEs
o gene deli e y in he CNS. The p ima y objec i e was o c ea e a
nanoca ie exhibi ing ex ensi e di usion in he b ain, ensu ing e ec-
i e deli e y and ans ec ion capaci y wi h di e en ypes o RNA.
3.1. De elopmen o ionizable nanoemulsions (iNEs)
To enhance b ain di usion, we designed a luid NE wi h a Vi amin E
co e combined wi h he ionizable lipid C12–200 [43]. Known o i s
cone-shaped s uc u e, C12–200 boos s endosomal dis up ion, c ucial
o e icien oligonucleo ide elease [10]. Upon cellula up ake, i be-
comes p o ona ed in acidic endosomes, igge ing a phase change ha
dis up s he endosomal memb ane, acili a ing ca go elease in o he
cy osol [44]. The combina ion o an oily co e wi h a lipid capable o
enhancing endosomal escape holds p omise o achie ing e ec i e b ain
di usion and modula ing CNS genes.
The ini ial sc eening o condi ions in ol ed bulk mixing, whe e
M.L. Bo ajo e al.
Jou nal o Con olled Release 372 (2024) 295–303
298
C12–200, DOPE, Vi amin E, and Tween 80® we e combined a a ious
mola a ios. This p elimina y in es iga ion led o he iden i ica ion o a
speci ic mola composi ion (35: 16: 46.5: 2.5) ha allowed a ela i ely
small pa icle size (165 nm, PDI 0.1) and a posi i e su ace cha ge (+20
mV). Once his mola composi ion was ixed, inc eased N/P a ios
(mola a io o ni ogen a oms in he complexing lipid o phospha e
a oms in he RNA) we e explo ed, esul ing in he iden i ica ion o he
ull en apmen o he RNA a N/P a io 15:1. The physicochemical
p ope ies o his ini ial o mula ion using di e en ypes o RNA can be
ound in Supplemen a y Table 1. Tween 80® was also subs i u ed by
DMG-PEG
2000
, his being a mo e ypical PEGyla ed ma e ial o RNA
deli e y.
Fu he mo e, he o mula ion p ocess unde wen adap a ion o
mic o luidic p epa a ion. The esul was a no able educ ion in pa icle
size, which was a ibu ed o he ine- uning o mixing pa ame e s
(speed and shape o he con e gence o bo h aqueous and o ganic phases
wi hin he de ice) [45,46]. Addi ionally, he p esence o he DMG-
PEG
2000
could also con ibu e o he educ ion in size o he esul ing
iNE [45,47].
To showcase he e sa ili y o he de eloped nanosys ems, a ious
ypes o RNAs we e encapsula ed in o he iNEs. The physicochemical
p ope ies o he esul ing iNEs, summa ized in Table 1, indica e ha ,
i espec i e o he ca go, he pa icle diame e , PDI, o su ace cha ge
a e e y simila . This consis ency illus a es he obus ness o he iNE o
deli e ing di e se oligonucleo ide ypes.
The encapsula ion e iciency and elease beha io we e assessed
h ough aga ose gel elec opho esis (Fig. 1), a e dilu ion o he iNEs in
di e en bu e s (ci a e bu e (CB) a pH 4, and phospha e bu e saline
(PBS) a pH 7.4). C12–200 exhibi s a posi i e cha ge a acidic pH, hus
acili a ing RNA condensa ion, and becomes neu al a physiological pH
[48–50]. The esul s in Fig. 1 indica e ha no ee-RNA and hence no
elease was obse ed upon dilu ion a pH 4 (CB condi ion) ega dless o
he RNA ype. A physiological pH (7.4, PBS condi ion), mos RNA
molecules emain complexed wi hin he iNE, necessi a ing T i on X-100
dis up ion o comple e elease.
3.2. In i o assessmen o iNEs wi h di e en PEGyla ed compounds in
HeLa cells
In i o expe imen s we e conduc ed using HeLa cells o assess he
cy o oxici y and ans ec ion e iciency o iNEs, employing siGFP as a
model RNA molecule. A compa ison was made be ween iNE o mula-
ions con aining Tween 80® (p epa ed by bulk mixing) and DMG-
PEG
2000
(p epa ed by mic o luidic mixing).
Rega ding cellula cy o oxici y (Fig. 2-A), iNE-DMG-PEG
2000
exhibi ed highe cell iabili y han iNE-Tween 80® a he highes con-
cen a ions (250 and 100 nM), displaying simila beha io a lowe
concen a ions (50 and 10 nM). On he o he hand, bo h iNE o mula-
ions showed compa able ans ec ion e iciency (pe cen age o ans-
ec ed cells and luo escence in ensi y), and, hus, simila p o ein
ansla ion p o iles a all concen a ions es ed (Fig. 2-B). Howe e , i
should be highligh ed ha a ela i ely low RNA concen a ions (50
nM), he ans ec ion e iciency and oxici y we e simila o bo h ypes
o o mula ions. Despi e hese ela i ely mino di e ences, he iNE
con aining DMG-PEG
2000
, p epa ed by mic o luidics, was selec ed o
subsequen s udies. O no e, he silencing e ec was e en obse ed a
e y low concen a ions o siRNA.
3.3. In i o e alua ion o iNEs in neu on, as ocy e, and mic oglia
cellula models
To e alua e he abili y o iNE nanosys ems o deli e oligonucleo-
ides o he b ain, in i o s udies we e conduc ed using human neu ons
Table 1
Physicochemical p ope ies o iNE de eloped, con aining C12–200, DOPE,
Vi amin E, and DMG-PEG, in combina ion wi h siGFP and mGFP.
Type o
RNA
Size
(nm)
PDI ζ-Po en ial
(mV)
Encapsula ion e iciency
(%)
siGFP 64 ±7 0.23 ±
0.03
(−)2 ±1 80–90
mGFP 72 ±9 0.17 ±
0.07
(−)4 ±2 80–90
Encapsula ion e iciency was measu ed by aga ose gel, and alues we e
co obo a ed by RiboG een assay. Abb e ia ions: GFP: g een luo escence p o-
ein. mGFP: mRNA encoding GFP. PDI: polydispe si y index. siGFP: siRNA an i-
GFP. Values ep esen he mean ±s anda d de ia ion (n ≥3).
Fig. 1. Encapsula ion e iciency o siGFP (le ) and mGFP ( igh ) loaded iNE.
Samples we e ea ed wi h CB (pH 4), PBS (pH 7.4), and T i on X-100 ( o iNE
dis up ion).
Abb e ia ions: CB: Ci a e bu e . GFP: G een luo escence p o ein. iNE: Ioniz-
able nanoemulsion. mGFP: mRNA encoding GFP. PBS: Phospha e-bu e ed sa-
line. siGFP: siRNA an i-GFP
Fig. 2. Cy o oxici y (A) and GFP silencing e ec (B) o iNE-Tween 80® (blue)
and iNE-DMG-PEG
2000
( ed) con aining siGFP, in HeLa cells exp essing GFP.
The silencing e ec was de e mined in e ms o he pe cen age o GFP nega i e
cells (B, le axis, ba s) and mean luo escence in ensi y (B, igh axis, symbols).
(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.)
Abb e ia ions: DMG-PEG
2000
: (R)-me hoxy-polye hyleneglycol-2000-ca ba-
moyl-di-O-my is yl-sn-glyce ide. GFP: G een luo escence p o ein. iNE: Ioniz-
able nanoemulsion. MFI: Mean luo escence in ensi y. PC: Posi i e con ol,
lipo ec amine. siGFP: siRNA an i-GFP. UT: Un ea ed. Values ep esen he
mean ±s anda d de ia ion (n ≥3)
M.L. Bo ajo e al.
Jou nal o Con olled Release 372 (2024) 295–303
299
(SH-SY5Y), a as ocy es (C6), and human mic oglia (CHME-3 cells) cell
models, wi h mGFP as he model nucleic acid (Fig. 3). No oxici y was
obse ed a any o he es ed concen a ions in he di e en cell models
(Fig. 3-A) when compa ed o un ea ed cells.
Rega ding he ans ec ion e iciency, signi ican le els o GFP-
ans ec ed cells we e de ec ed in all h ee cellula models, each
exhibi ing o e 70% GFP-posi i e cells (Fig. 3-B). The sligh ly highe
le els o ans ec ion obse ed in C6 cells compa ed o CHME-3 cells
could be a ibu ed o g ea e cellula up ake by as ocy es o e
mic oglia cells. On he o he hand, ega ding he luo escence in ensi y
alues, iNE-mGFP esul ed in signi ican ly inc eased le els o bo h
neu ons and mic oglia models (Fig. 3-C). Howe e , a signi ican ly lowe
ans ec ion e iciency was obse ed o as ocy es, whe e no signi ican
di e ences we e ound when compa ed wi h he un ea ed cells. A
plausible explana ion o his obse a ion could be ela ed o a lowe
up ake capaci y o dis inc in acellula a icking o as ocy es as
compa ed o he neu onal and mic oglia cells.
3.4. In i o e alua ion o CNS di usi i y o iNEs encapsula ing mGFP
Wi h he aim o simul aneously e alua ing di usi i y and unc ional
pe o mance, iNEs loaded wi h mGFP we e di ec ly adminis e ed in he
b ain, ollowing in a-pa enchymal (in a halamic) injec ion, in a s.
Fig. 4 shows he ans ec ion in neu ons, as ocy es, and mic oglia a 24
h pos -adminis a ion. Neu on analysis equi ed a double immuno-
labelling p ocess due o inhe en luo escence in neu ons (Supplemen-
a y Fig. 1). The images exhibi ed dis inc GFP signals in neu ons,
indica ing he up ake o iNEs by his cell ype and he subsequen
ansla ion o he epo e p o ein. Mo eo e , a speci ici y o iNEs o
neu ons s mic oglia and as ocy es was no ed (Fig. 4). In e ms o
mic oglia up ake, hese indings con as wi h ou p e ious in i o
luo escence in ensi y esul s (Fig. 3-B,C), indica ing a lack o co ela-
ion be ween in i o-in i o ou comes, ypically obse ed wi h nano-
pa icles [51,52]. These esul s imply he po en ial o iNEs o e ec i ely
e ade mic oglia cells, known as he esiden mac ophages o he CNS
[53,54].
O e all, hese esul s sugges ha ans ec ion o speci ic b ain cells
could be dependen on he iden i y o he nanopa icle i sel . In ag ee-
men wi h his, classical LNPs e icien ly we e epo ed o ans ec
neu ons, as ocy es and, o a mino ex en , he mic oglia cells, upon
di ec adminis a ion in he b ain [55]. Good ans ec ion alues in
neu ons and as ocy es we e also epo ed o LNPs con aining hiol-
clea able lipids, upon in ace eb o en icula (ICV) adminis a ion
[17]. O no e, mic oglia cells we e no included in his s udy. As a
di e en deli e y app oach, o he au ho s ha e ound ha poly-
cap olac one-based nanopa icles could be designed o be di ec ed
speci ically o mic oglia cells [56]. Despi e hese s udies, as o now, no
clea explana ion o he di e en cellula p e e ences o di e en
nanopa icles has been iden i ied. We hypo hesize ha he composi ion
o he nanopa icles in luences he esul ing p o ein co ona a ound he
nanoca ie s, leading o di e en cellula up ake and e en in acellula
a e.
The high speci ici y o neu ons unde sco es he po en ial o ou iNEs
o he ea men o b ain diseases ha could bene i om di ec CNS
adminis a ion, such as glioblas oma o Pa kinson’s disease [57–60].
Howe e , i is essen ial o in e p e his conclusion cau iously, consid-
e ing ha biodis ibu ion may be signi ican ly di e en by he in luence
o hese disease condi ions.
Following he assessmen o he ans ec ion e iciency o iNE, he
di usi i y o he nanoca ie s h oughou he en i e b ain o he animals
was in es iga ed (Fig. 5). In e es ingly, a no able di usion was obse ed
om he injec ion si e, as indica ed by a ed GFP signal, o mo e dis an
a eas wi hin he same hemisphe e. This obse a ion is ema kable i we
conside he limi ed li e a u e e alua ing he di usion o mRNA-loaded
nanopa icles in he b ain. In a ecen s udy, adi ional LNPs ca ying
C e mRNA we e di ec ly injec ed in o he s ia um and hippocampus o
animals. This led o gene edi ing away om he injec ion si e, wi h a
di usion ange o 1.2–2.7 mm, depending on he si e o injec ion and he
dose adminis e ed [55]. These esul s a e suppo ed by o he mRNA-
con aining LNPs adminis e ed ICV, leading o 1–1.5 mm di usion pa -
e ns om he injec ion si e [15,16]. Ou iNE e ealed cells exp essing
GFP up o app oxima ely 3.4 mm apa , based on luo escen exp ession
on close-up images (Supplemen a y Fig. 2). This alida es ou hypo h-
esis ha so luid co es could enhance he di usion o nanoca ie s.
Ne e heless, i is c ucial o no e ha he si e and a e o adminis a ion
may also impac he di usion pa e n o he nanopa icles, and ha
u he di usion measu emen s would be needed o be e unde s and
he dis ibu ion pa e n o ou iNEs.
Close-up images u he con i med he colocaliza ion o he luo-
escence p o ein in neu ons and we e used o es ima e he di usion
dis ance o ou iNEs (Supplemen a y Fig. 2). The speci ici y o he signal
was con i med by he absence o luo escence upon PBS adminis a ion
(Supplemen a y Fig. 3).
O e all, based on he obse ed dis ibu ion p o ile, we ha e hy-
po hesized ha he di usion o ou iNEs is in luenced by he oily co e o
he iNE nanoca ie . P e ious wo k in ou g oup wi h polyaminoacid
nanocapsules (con aining an oily co e) showed issue di usi i y
ollowing subcu aneous adminis a ion could be enhanced by unning
pa icle diame e (a ound 100 nm) and su ace nanoca ie composi ion
[34,61]. In his ega d, based on p e ious epo s, we could a gue ha
he PEG componen s o he su ace o iNEs could enhance di usi i y
[30–32].
These indings unde sco e he po en ial o iNEs o di use o a ious
b ain a eas and e icien ly ans ec mRNA speci ically in o neu ons,
u he con i ming he ansduc ion o he desi ed model p o ein. The
Fig. 3. Cy o oxici y (A) and GFP ans ec ion e iciency (B–C) o iNE-mGFP
(b igh colou ) in a neu on ( ed), as ocy e (g een), and mic oglia (blue)
cellula models. T ans ec ion e iciency was de e mined in e ms o he pe -
cen age o GFP-posi i e cells (B) and mean luo escence in ensi y (C). Com-
pa ison was made wi h un ea ed cells (pale colou ). (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.)
Abb e ia ions: GFP: G een luo escence p o ein. iNE: Ionizable nanoemulsion.
MFI: Mean luo escence in ensi y. mGFP: mRNA encoding GFP. UT: Un ea ed.
A signi ican compa ison was pe o med using an o dina y one-way ANOVA
ollowed by Tukey’s mul iple compa ison es s be ween g oups. P- alues <0.05
we e conside ed s a is ically signi ican (*). Also, (****) i p- alue <0.0001. Ns:
No signi ica i e. Values ep esen he mean ±s anda d de ia ion (n ≥3)
M.L. Bo ajo e al.

Jou nal o Con olled Release 372 (2024) 295–303
300
esul s sugges ha iNE can acili a e he deli e y o gene ic ca go o
di e en b ain egions ollowing a single adminis a ion.
4. Conclusions
He e, a new RNA deli e y ca ie named iNE, exhibi ing a unique
p o ile as compa ed o he classical LNPs, is disclosed. The iNEs
exhibi ed sub-100 nm size, neu al su ace cha ge, high RNA en apmen
capaci y, and a o able ans ec ion p o ile in di e en cell lines in i o,
no ably in neu onal and mic oglia cells. While hese p ope ies may be
common o o he nanoca ie s, iNEs showed an ad an ageous p o ile
ma ked by hei high di usi i y ac oss he b ain issue upon hei di ec
adminis a ion in a pa enchyma.
In summa y, his esea ch ou lines he de elopmen o a no el class
o ionizable nanoca ie s ailo ed o RNA deli e y o he b ain, show-
casing a p omising di usion pa e n and speci ic a ge ing capabili ies
o neu ons. These nanoca ie s hold signi ican p omise o add essing
di e se CNS diseases, including glioblas oma o Pa kinson’s disease.
Fig. 4. Immunolabelling o GFP in neu ons (labeled wi h βIII- ubulin), as ocy es (labeled wi h GFAP), and mic oglia (labeled wi h Iba-1) cells in a s a e
in apa enchymal adminis a ion o con ol (PBS) o iNE-mGFP. Hoechs was used as nuclei ma ke .
Abb e ia ions: βIII- ubulin: Class III β- ubulin. GFAP: Glial ib illa y acidic p o ein. GFP: G een luo escence p o ein. Iba-1: Ionized calcium-binding adap o molecule
1. iNE: Ionizable nanoemulsion. mGFP: mRNA encoding GFP
M.L. Bo ajo e al.
Jou nal o Con olled Release 372 (2024) 295–303
301
CRediT au ho ship con ibu ion s a emen
Mi eya L. Bo ajo: Concep ualiza ion, Da a cu a ion, Fo mal anal-
ysis, In es iga ion, Me hodology, Valida ion, W i ing – o iginal d a ,
W i ing – e iew & edi ing, Visualiza ion. Aloia Quijano: Fo mal
analysis, In es iga ion, Me hodology, Valida ion, Visualiza ion. Philipp
Lapuhs: Fo mal analysis, In es iga ion, Me hodology, Valida ion,
W i ing – e iew & edi ing. Ana I. Rod iguez-Pe ez: In es iga ion,
Me hodology, Valida ion. Shubaash An hiya: Concep ualiza ion,
In es iga ion, Me hodology, W i ing – e iew & edi ing. Jos´
e L. Lab-
andei a-Ga cia: Concep ualiza ion, Funding acquisi ion, Resou ces,
Supe ision, W i ing – e iew & edi ing. Ri a Valenzuela: Concep u-
aliza ion, Da a cu a ion, Fo mal analysis, In es iga ion, Me hodology,
P ojec adminis a ion, Supe ision, Valida ion, Visualiza ion, W i ing –
o iginal d a , W i ing – e iew & edi ing. Ma ía Jos´
e Alonso:
Concep ualiza ion, Da a cu a ion, Funding acquisi ion, P ojec admin-
is a ion, Resou ces, Supe ision, W i ing – o iginal d a , W i ing –
e iew & edi ing.
Da a a ailabili y
Da a will be made a ailable on eques .
Acknowledgmen s
This wo k was suppo ed by B-SMART p ojec , unded by he Eu o-
pean Union’s Ho izon 2020 esea ch and inno a ion p og am unde
g an ag eemen No 721058. Au ho s also acknowledge he Compe i i e
Re e ence G oups, Conselle ía de Educaci´
on e O denaci´
on Uni e si a ia,
Xun a de Galicia (Re . ED431C 2021/17 and ED431C 22/41). Financial
suppo om he Xun a de Galicia (Cen o singula de In es igaci´
on de
Galicia acc edi a ion 2019-2022), he (Eu opean Union Eu opean
Regional De elopmen Fund – ERDF), and he Spanish Minis y o Sci-
ence and Inno a ion (PID2021-126848NB-100) a e g a e ully
acknowledged. Mi eya L. Bo ajo acknowledges he inancial suppo by
Ins i u o de Salud Ca los III h ough he “Con a os i-PFIS: Doc o ados
ISS-emp esa en Ciencias y Tecnologías de la Salud” (IFI19/00033). The
au ho s also wan o acknowledge Mu hiah Manoha an, om Alnylam
Pha maceu icals (MA, USA) o gene ously p o iding C12-200 and
DMG-PEG
2000
lipids o ou expe imen s. G aphical abs ac was c ea ed
wi h BioRende .com.
Appendix A. 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.2024.06.051.
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