Engineering, on-demand manufacturing, and scaling-up ofpolymeric nanocapsules

RESEARCH REPORT
Enginee ing, on-demand manu ac u ing, and scaling-up o
polyme ic nanocapsules
José C ecen e-Campo | Ma ía José Alonso
Cen e o Resea ch in Molecula Medicine
and Ch onic Diseases (CIMUS), Campus Vida,
Uni e sidade de San iago de Compos ela,
San iago de Compos ela, Spain
Co espondence
Ma ía José Alonso, Cen e o Resea ch in
Molecula Medicine and Ch onic Diseases
(CIMUS), Campus Vida, Uni e sidade de
San iago de Compos ela, San iago de
Compos ela, 15782, Spain.
Email: [email p o ec ed].
Funding in o ma ion
Cen e o Indus ial Technological
De elopmen . Minis e io de Economía y
Compe i i idad. Axencia Galega de inno ación,
G an /Awa d Numbe : COLIVAC-FEDER
INNTERCONECTA-2012-CE277; Conselle ía
de Cul u a, Educación e O denación
Uni e si a ia, Xun a de Galicia; G upos de
e e encia compe i i a, G an /Awa d Numbe :
ED431C 2017/09
Abs ac
Polyme ic nanocapsules a e e sa ile deli e y sys ems wi h he capaci y o load lipophilic d ugs
in hei oily nucleus and hyd ophilic d ugs in hei polyme ic shell. The objec i e o his wo k
was o expand he echnological possibili ies o p epa e cus omized nanocapsules. Fi s , we
adap ed he sol en displacemen echnique o modula e he pa icle size o he esul ing nano-
capsules in he 50–500 nm ange. We also p oduced nanosys ems wi h a shell made o one o
mul iple polyme laye s i.e. chi osan, dex an sulpha e, hyalu ona e, chond oi in sulpha e, and
algina e. In addi ion, we iden i ied he condi ions o ansla e he p ocess in o a minia u ized
high- h oughpu ailo -made ab ica ion ha enables massi e sc eening o o mula ions. Finally,
he p oduc ion o he nanocapsules was scaled-up bo h in a ba ch p oduc ion, and also using
mic o luidics. The e sa ili y o he p ope ies o hese nanocapsules and hei ab ica ion ech-
nologies is expec ed o p opel hei ad ance om bench o clinic.
KEYWORDS
high- h oughpu sc eening, laye -by-laye , mic o luidics, nanocapsules, pa icle size, scale-up
1|INTRODUCTION
Nanocapsules (NCs) a e nanome ic sys ems wi h an inne co e and
an ex e nal shell. Depending on hei composi ion, NCs ha e been
named as lipid NCs, consis ing o an oily co e s abilized by PEGyla ed
amphiphilic molecules; and polyme ic NCs, when he oily nucleus is s abi-
lized by a polyme ic shell. Al-Kou i e al. desc ibed in 1986 he i s poly-
me ic NCs, made o polyisobu ylcyanoac yla e (PACA).
1
In 1989, Fessi
e al. epo ed he p epa a ion o poly-(D,L-lac ide) (PLA) NCs by he
sol en -displacemen echnique.
2
Subsequen ly, ou g oup ex ended he
applica ion o his echnology o he p oduc ion o NCs wi h a
hyd ophobic shell, ha is, poly-ε-cap olac one (PCL) NCs,
3–5
and a a i-
e y o hyd ophilic polyme shells consis ing o chi osan (CS),
6
hyalu onic
acid,
7
poly-L-aspa agine,
8
polyglu amic acid,
9
polya ginine,
10
and
p o amine.
11
Due o hei lipidic co e, NCs we e, o iginally, concei ed as sui -
able ca ie s o lipophilic d ugs. Howe e , ou lab has expanded his
echnology o allow NCs o ca y hyd osoluble mac omolecules, such
as p o eins,
12,13
pep ides,
14–16
and polynucleo ides,
10
using hem in
di e en he apeu ic a eas. Fo example, in he oncology ield, we
ha e de eloped di e en polyme ic NCs con aining he cy o oxic
d ugs pli idepsin and doce axel,
10,17–21
which we e able o p olong
he blood ci cula ion ime o hese d ugs and educe hei oxici y. In
addi ion, an impo an accumula ion o he d ugs in he lympha ic sys-
em was obse ed. In he accinology ield, we ha e success ully associ-
a ed di e en ypes o p o ein an igens, such as e anus oxoid,
13
in luenza an igen,
22
ecombinan hepa i is B su ace an igen,
23
and Iu A
E. coli an igen
24
o di e en NCs. The o e all esul obse ed, when
using hese an igens, was an inc eased immunogenic esponse ollowing
ei he , in amuscula o in anasal immuniza ion. Finally, we ha e also
Abb e ia ions: Alg, Algina e; ChS, Chond oi in sulpha e; CM-β-glucan, Ca boxy-
me hyl-β-glucan; CS, Chi osan; CTAB, Hexadecyl ime hylammonium b omide;
DLS, Dynamic ligh sca e ing; DS, Dex an sulpha e; FESEM, Field emission
scanning elec on mic oscopy; HA, Hyalu ona e; HTS, High- h oughpu sc een-
ing; LbL, Laye -by-laye ; Lec, leci hin; Mw, Molecula weigh ; NC, Nanocapsule;
NE, Nanoemulsion; P407, poloxame 407; PACA, Polyisobu ylcyanoac yla e;
PCL, Poly-ε-cap olac one; PDI, Polydispe si y index; PLA, Poly-(D,L-lac ide);
STEM, Scanning ansmission elec on mic oscope; T80, Tween 80; TPGS, D-
α-Tocophe ol polye hylene glycol 1,000 succina e
Recei ed: 11 June 2018 Re ised: 4 Sep embe 2018 Accep ed: 9 Sep embe 2018
DOI: 10.1002/b m2.10118
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium,
p o ided he o iginal wo k is p ope ly ci ed.
© 2018 The Au ho s. Bioenginee ing & T ansla ional Medicine published by Wiley Pe iodicals, Inc. on behal o The Ame ican Ins i u e o Chemical Enginee s.
38 wileyonlinelib a y.com/jou nal/b m2 Bioenginee ing & T ansla ional Medicine. 2019;4:38–50.
ound ha polyme ic NCs can inc ease he bioa ailabili y o di e en
d ugs adminis e ed h ough di e en mucosal ou es. Fo example, ou
esul s ha e shown he possibili y o inc easing he co neal pene a ion
o d ugs associa ed o he NCs.
6,25
Simila ly, we ha e ound ha poly-
me ic NCs led o an enhancemen o he sys emic abso p ion o pep ide
and p o ein d ugs adminis e ed by he o al
11,15,16
o nasal
14,26
ou es.
Apa om hese uses, and due o hei e sa ile na u e,
NCs ha e also been loaded wi h compounds wi h di e en
ac i i ies: an i-in lamma o y,
27–29
an ibac e ial,
30,31
an i ungal,
32–35
an ioxidan ,
28,36,37
and immunosupp essan ,
38
among many o he s.
39
Besides he sol en -displacemen echnique, wo o he widely
used NCs p epa a ion me hods a e he high ene gy-homogeniza ion
and he phase in e sion empe a u e me hod.
40–43
In gene al, i is
known ha he a io be ween he di e en phases (sol en and
nonsol en ),
39
he ype o oil and polyme s used, and hei ela i e
concen a ion
44
may in luence he size and su ace p ope ies o NCs
ob ained by sol en displacemen . Howe e , he in luence o o he
echnological pa ame e s, such as he way and a e o mixing o he
wo phases has no been sys ema ically in es iga ed. A be e unde -
s anding o he impac o hese a iables would allow a mo e p ecise
con ol o e he pa icle size when enginee ing NCs. On he o he
hand, a combina ion o he sol en -displacemen echnique wi h he
laye -by-laye app oach o e s in e es ing possibili ies o modi y he
NCs su ace p ope ies and, hence, o in luence he s abili y o he
NCs and hei in e ac ion wi h he biological sys ems. Mo eo e , he
na u e o he mul iple shell polyme s may acili a e he loading o di -
e en d ugs, and hei con olled elease.
24,45,46
Taking all his in o conside a ion, ou goal in his s udy was o
design o mula ion and echnological app oaches o p oduce ailo -
made polyme ic NCs, and o do so acco ding o di e en scale-down
(mic oli e s) and scale-up (li e ) echniques. The knowledge gene a ed
du ing hese s udies will hope ully con ibu e o a mo e s aigh o -
wa d ansla ion o polyme ic NCs om bench o clinic.
2|MATERIALS AND METHODS
2.1 |Ma e ials
2.1.1 |Oils
DL-α-Tocophe ol (Calbiochem) and squalene (densi y: 0.855 g/mL)
we e ob ained om Me ck Millipo e (Da ms ad , Ge many). Miglyol
812 was kindly gi ed by C eme Oleo GmbH & Co. KG (Hambu g,
Ge many).
2.1.2 |Su ac an s
Deoiled phospha idylcholine-en iched L-α-leci hin (Epiku on 145 V)
was a gi om Ca gill (Ba celona, Spain). Poloxame 407 (Plu onic
127) was ob ained om BASF (Ludwigsha en, Ge many). D-
α-Tocophe ol polye hylene glycol 1,000 succina e (TPGS) was
pu chased o An a es Heal h P oduc s Inc. (Jonesbo ough, TN).
Polye hylene glycol so bi an monoolea e (Tween 80), hexadecyl i-
me hylammonium b omide (CTAB), and sodium chola e hyd a e
we e pu chased om Sigma-Ald ich (S . Louis, MO).
2.1.3 |Polyme s
Ul apu e CS hyd ochlo ide sal (P o asan UP CL 113, Mw 125 kDa,
deace yla ion deg ee o 86%) was pu chased om No ama ix
(Sand ika, No way). Dex an sul a e sodium sal (Mw 6–8 kDa) was
bough o MP Biomedicals (Illki ch, F ance). Fo he p epa a ion o he
NCs in he 96-well pla e pha maceu ical g ade CS hyd ochlo ide wi h
a Mw o 47 kDa and a 80–95% deace yla ion deg ee was acqui ed
om Heppe Medical Chi osan GmbH (Halle, Ge many), and dex an
sul a e sodium sal pha maceu ical g ade, wi h a Mw 8 kDa was pu -
chased om Sigma-Ald ich SAFC (Madison, WI). Polya ginine
(Mw 29 kDa was ob ained om PTS (Valencia, Spain). Sodium algina e
ULV-L3 ( iscosi y 10% solu ion 27 mPas) was pu chased om Kimica
Co po a ion (Tokyo, Japan). Ca boxyme hyl-β-glucan sodium sal ,
ob ained om Saccha omyces ce e isae and modi ied wi h ca boxy-
me hyl g oups a an 85% subs i u ion deg ee, was a kind dona ion
om Mibelle AG Biochemis y (Buchs, Swi ze land). Sodium hyalu o-
na e (HA; Mw 57 kDa) was pu chased om Li eco e Biomedical
(Chaska, MN). Poly-L-glu amic acid sodium sal (Mw 15–50 kDa), and
chond oi in-6-sulpha e sodium sal we e ob ained om Sigma-
Ald ich.
2.1.4 |O he s
All o he chemicals used we e o eagen g ade o highe pu i y.
2.2 |Design o expe imen s
The S a g aphics Cen u ion XVI.I so wa e was used o design he
expe imen s. Two esponse a iables (size and polydispe si y index
[PDI]) and h ee expe imen al ac o s: (a) a e o addi ion o he
o ganic phase o e he aqueous phase (pou ing s. injec ion),
(b) olume o he o ganic phase (e hanol olume om 0.25 o 5 mL),
and (c) olume o aqueous phase ( om 5 o 15 mL) we e speci ied. A
ac o ial 2
3
design was pe o med, wi h ou cen e poin s pe block
and a andom cen e poin placemen . The selec ed design had 12 uns,
wi h one sample o be aken each un. The de aul model was 2- ac o
in e ac ions wi h se en coe icien s.
Rega ding he expe imen al p ocedu e, he NCs we e p epa ed by
he sol en -displacemen echnique, using a modi ica ion o he
me hod p e iously de eloped by us.
47
B ie ly, an o ganic phase con-
aining 30 mg o i amin E and 10 mg o leci hin in he equi ed
amoun o e hanol was added o e an aqueous phase con aining 5 mg
o CS in he equi ed amoun o wa e . A e he addi ion o he
o ganic phase o e he aqueous phase by ei he he pou ing o he
injec ing p ocedu e he sample was s i ed a 300 pm o 10 min.
Finally, wi hou emo ing he sol en , size and PDI we e measu ed by
dynamic ligh sca e ing (DLS).
2.3 |NCs p epa a ion by ei he pou ing o injec ing
he o ganic phase o e he aqueous phase
The o ganic phase o hese o mula ions consis ed o 0.5 mL solu ion
o he oil (60 mg/mL) and 0.5 mL o he su ac an (20 mg/mL), bo h
in e hanol. In hose cases whe e a co-su ac an was included, 25 μlo
an aqueous solu ion o his componen (sodium chola e 200 mg/mL
o CTAB 66.67 mg/mL) we e added. Finally, he olume was adjus ed
CRECENTE-CAMPO AND ALONSO 39
wi h pu e e hanol up o 2.5 mL. The aqueous phase was p epa ed dis-
sol ing 5 mg o he polyme in 10 mL o ul apu e wa e , o jus wa e
o nanoemulsions. The addi ion o he o ganic phase o e he aque-
ous phase was made in wo di e en ways: by pou ing one phase o e
he o he o by injec ing he o ganic phase inside he aqueous phase
h ough a needle (100 S e ican, 0.60 ×60 mm, 23G ×2
3/8
00, B aun,
Melsungen, Ge many) applying high manual p essu e. In bo h cases,
he aqueous phase was main ained unde s i ing du ing he addi ion.
A e 10 min o agi a ion a 300 pm, samples we e cha ac e ized
by DLS.
2.4 |P epa a ion o la ge sizes NCs
The sol en -displacemen echnique was eadjus ed o modula e he
NCs pa icle size. Thus, 0.5 mL o a solu ion o i amin E 60 mg/mL
and 0.5 mL o a solu ion o leci hin 20 mg/mL, bo h in e hanol, we e
mixed in a es ube. Upon s i ing a 700 pm, an ini ial olume o
wa e was added wi h a mic opipe e. The emulsion was main ained
unde hese condi ions o a speci ic ime. A e ha ime, 4 mL o an
aqueous solu ion o CS 1.25 mg/mL we e added, and he suspension
s i ed o 10 min. Finally, he NCs we e cha ac e ized by DLS.
2.5 |Laye -by-laye coa ing o NCs
Di e en olumes o NCs we e placed in glass HPLC ials. To hese
samples, and unde s i ing a 300 pm, di e en olumes o a second
polyme solu ion we e added up o a o al o 1 mL, while keeping he
a io polyme laye 1:polyme laye 2 (w/w) cons an . The mix u e
was s i ed o 30 min and cha ac e ized by DLS. Once he a io o
olumes was selec ed, a ixed olume o NCs was placed in a glass
HPLC ial. To his solu ion, and unde s i ing a 300 pm, a ixed ol-
ume o a second polyme solu ion was added up o 1 mL, es ing di -
e en a ios polyme laye 1:polyme laye 2 (w/w). The mix u e was
s i ed o 30 min and hen cha ac e ized by DLS. This p ocedu e was
epea ed o each consecu i e laye .
2.6 |NCs p epa a ion by a high- h oughpu
sc eening-adap able p ocedu e
NCs composed o di e en ma e ials (Suppo ing In o ma ion
Table S1) we e p epa ed adap ing he sol en -displacemen me hod
o a 96-mul iwell pla e. B ie ly, an o ganic phase was p epa ed mixing
50 μl o a 72 mg/mL e hanolic solu ion o he selec ed oil, 40 μlo a
37.5 mg/mL e hanolic solu ion o he su ac an , and 10 μl o he co-
su ac an solu ion (i needed). As a co-su ac an an aqueous solu ion
o sodium chola e 30 mg/mL o CTAB 10 mg/mL we e used o posi-
i e and nega i ely cha ged NCs, espec i ely. Fo he NEs and he
combina ions con aining a posi i e polyme and leci hin, o a posi i e
polyme and squalene, he co-su ac an was no included. The
o ganic phase was pou ed wi h a mic opipe e in o he co esponding
well o a 96-mul iwell pla e con aining 200 μl o 1.5 mg/mL aqueous
solu ion o he polyme o jus wa e in he case o NEs. The addi ion
was made unde ho izon al shaking (300 pm) and samples we e incu-
ba ed o 10 min be o e cha ac e iza ion by DLS.
2.7 |P epa a ion o NCs wi h di e en olumes o
e hanol
Abou 30 mg o i amin E and 10 mg o leci hin we e dissol ed in di -
e en olumes o e hanol. This solu ion was added o e 10 mL o an
aqueous solu ion o CS (0.5 mg/mL). The addi ion was done by ei he
pou ing he o ganic phase o e he aqueous phase o by injec ing i
h ough a needle (100 S e ican, 0.60 ×60 mm, 23G ×2
3/8
00, B aun,
Melsungen, Ge many) applying high manual p essu e. In bo h cases,
he aqueous phase was main ained unde s i ing. A e 10 min o agi-
a ion a 300 pm, he excess o e hanol was emo ed using a o a y
e apo a o (Büchi, Swi ze land) and he olume was adjus ed o 5 mL
wi h ul apu e wa e .
2.8 |Ba ch scale-up o NCs
Fo a 100 mL ba ch, an o ganic solu ion was p epa ed mixing 2 mL o
leci hin 50 mg/mL and 0.5 mL o i amin E 600 mg/mL bo h in e ha-
nol. The inal olume was adjus ed wi h e hanol o 10 mL. This solu-
ion was pou ed in o 100 mL o an aqueous solu ion o CS 0.5 mg/mL
unde agi a ion a 500 min
−1
, using a p opelle s i e IKA RW
20 (S augen, Ge many; 4-bladed, s i diame e 50 mm, sha diame e
8 mm, and sha leng h 350 mm).
Fo a 1 L ba ch, an o ganic solu ion con aining 1 g o leci hin and
3 g o i amin E in 100 mL o e hanol was p epa ed. This solu ion was
pou ed in o 1 L o an aqueous solu ion o CS 0.5 mg/mL unde agi a-
ion a 500 min
−1
, using a p opelle s i e IKA RW 20 (S augen, Ge -
many; 4-bladed, s i diame e 10 cm, sha diame e 8 mm, and sha
leng h 350 mm). A e 10 min o s i ing sample was cha ac e ized
by DLS.
2.9 |NCs p epa a ion by mic o luidics
A NanoAssembl Bench op (P ecision nanosys ems, Vancou e ,
Canada) sys em was used o p epa e he NCs. The ca idge channels
dimensions we e 200 μm wide and 79 μm high, wi h he ingbone
s uc u es o med by 31 μmhighand50μm hick in he mixe a ea. To
check he in luence o he o al low a e his low was a ied om 2.5
o 15 mL/min. The o ganic phase was a mix u e o 0.5 mL o i amin E
60 mg/mL and 0.5 mL o leci hin 20 mg/mL, bo h in e hanol. The aque-
ous phase was p epa ed by dissol ing 5 mg o CS in 9 mL o wa e . The
o al concen a ion in his case was 4.5 mg/mL. To check he in luence
o he componen s concen a ion, a o al low a e o 10 mL/min was
selec ed. While he a io o he componen s was cons an , hei con-
cen a ion a ied om 2.25 mg/mL o 22.50 mg/mL. A e he samples
we e p epa ed, hei pa icle size and PDI was cha ac e ized by DLS.
2.10 |Physicochemical cha ac e iza ion
Pa icle size and polydispe si y index by DLS using a Ze asize Nano-S
(Mal e n Ins umen s; Mal e n, UK). Ze a po en ial was de e mined
by lase Dopple anemome y, using he same equipmen . I no indi-
ca ed, analyses we e pe o med a 25 C wi h a de ec ion angle o
173in dis illed wa e .
The mo phology o he NCs was examined by ield emission scan-
ning elec on mic oscopy (FESEM; ZEISS, ULTRA Plus, Ge many). Fo
40 CRECENTE-CAMPO AND ALONSO
he analysis, he NCs we e dilu ed in wa e 1:1000 and mixed wi h
he same olume o 2% (w/ ) phospho ungs ic acid solu ion. A olume
o 1 μl o his mix u e was placed on coppe g ids wi h ca bon ilms.
The g ids we e le o d y in he open ai and hen hey we e washed
wi h 1 mL o wa e . Once he g ids we e d ied hey we e obse ed in
he mic oscope using bo h STEM and imme sion lens (In-Lens)
de ec o s.
2.11 |S a is ical analysis
Unless o he wise indica ed, he expe imen s we e epea ed a leas
h ee imes. The esul s a e p esen ed as mean SD. Fo he compa -
ison o he NCs pa icles sizes by pou ing o injec ing he o ganic
phase o e he aqueous phase (sec ion 3.1.1) a mul iple es was pe -
o med meanwhile o he compa ison o he pa icles sizes in
sec ion 3.1.2 a one-way ANOVA was pe o med. The di e ences we e
conside ed signi ican o *p< .05, ** p< .01, *** p< .001, and ****
p< .0001. All he s a is ical analyses we e ca ied ou wi h G aphPad
P ism Ve sion 6.0 so wa e (G aphPad so wa e Inc., La Jolla, CA).
3|RESULTS AND DISCUSSION
Polyme ic NCs can be easily p epa ed using he sol en -displacemen
echnique (Suppo ing In o ma ion Figu e S1). The lipids a e dissol ed
in a sol en phase (also e e ed o as o ganic phase), which spon ane-
ously o ms NCs when pou ed in o a nonsol en phase (usually an
aqueous phase). Molecules ha we e soluble in he sol en phase
exceed hei he modynamic solubili y limi when he sol en and
nonsol en phases mixes, which esul s in he o ma ion o oily nano-
d ople s. Simul aneously, he polyme solubilized in he aqueous
phase ge s adso bed on o he oily d ople s su ace due o i s elec o-
s a ic in e ac ion wi h su ac an s o opposi e cha ge. Finally, he
excess o o ganic sol en , i any, is elimina ed by e apo a ion.
F om a echnological poin o iew, i is c ucial o ha e e sa ile
me hods ha enable he con ol o he nanosys em’s size, as his
p ope y is known o in luence hei in e ac ion wi h he biological
sys ems.
48–50
Mo eo e , om a quali y con ol poin o iew, he
accu acy o his pa ame e is essen ial o he high scale p oduc ion
and he clinical de elopmen o a o mula ion. Ha ing his in mind, in
his s udy, we ha e s udied sys ema ically he in luence o he compo-
si ion and echnological pa ame e s on he size o NCs p epa ed by
he sol en -displacemen echnique.
3.1 |Modi ica ion o he NCs pa icle size
3.1.1 |P epa a ion o NCs wi h pa icle size <100 nm
The p oduc ion o nanosys ems wi h pa icle sizes smalle han
100 nm may be o in e es o speci ic applica ions. Fo example, in
he a ea o oncology, a small size is c i ical o imp o ing he pene a-
ion ac oss he umo .
51,52
Simila ly, in accinology, he small size may
a o he lympha ic d ainage.
53,54
P e ious s udies by ou esea ch g oup showed ha he dilu ion
o Miglyol 812 and leci hin in he o ganic phase, and he d opwise
addi ion o his phase o e an aqueous one lead o a signi ican
dec ease in he a e age size o he ob ained oily nanod ople s, om
~200 o 100 nm.
55
Based on his esul , we adop ed an expe imen al
design in which we kep cons an he mass and a io o he NCs com-
ponen s, and we a ied he ollowing pa ame e s: (a) he olume o
he o ganic phase (e hanol), (b) he olume o he aqueous phase, and
(c) he way he o ganic phase was added o e he aqueous phase
(pou ing s. injec ing). As a s anda d NC composi ion, we chose one
p e iously epo ed by ou g oup ha consis ed on a combina ion o
CS and i amin E wi h leci hin as a su ac an .
24
The esponse su ace,
p esen ed in Figu e 1, shows ha he smalles pa icle sizes we e
ob ained wi h he highes olumes o e hanol (5 mL) and wa e
(15 mL), con i ming ha , as expec ed, he dilu ion o lipophilic and
hyd ophilic componen s in hei espec i e phases a o s he o ma-
ion o smalle pa icle sizes. On he o he hand, he injec ion o he
o ganic phase inside he aqueous phase h ough a needle has a clea
impac in he NCs pa icle size when compa ed wi h he echnique
consis ing on jus pou ing one phase o e he o he , e en when he
concen a ion o he s a ing solu ions emains he same. The addi-
ion, in bo h cases, is qui e as .
The molecula mechanisms behind he nanodispe sion achie ed
by he sol en -displacemen echnique ha e been a ibu ed o in e -
acial u bulences be ween he sol en and nonsol en phases, known
as he Ma angoni e ec , which leads o he sepa a ion o he sys em
in o wo phases.
56,57
O he au ho s a ibu e he o ma ion o he
NCs o a simul aneous p ocess o emulsi ica ion d i en by he “ouzo
e ec ”
58,59
and polyme deposi ion o e he oily nanod ople s. In he
case o polyme ic NCs, his means ha he local supe sa u a ion o
he oil d i es a spon aneous nuclea ion in he o m o small oily nano-
d ople s. The al eady o med nuclei g ow by agg ega ion o by di u-
sion o oil molecules om he su oundings. The g ow h con inues
un il he oil concen a ion eaches he equilib ium sa u a ion concen-
a ion.
60
Expe imen ally, o ob ain small pa icles sizes, we should
a o apid nuclei o ma ion and li le o no pa icle g ow h. This can
be achie ed, as indica ed in Figu e 1, by dec easing he concen a ion
o he oil, o a oid pa icle g ow h, and/o injec ing he nonsol en
in o he sol en phase, which p oduces u bulences ha c ea e mul i-
ple small nuclei o oily nanod ople s.
Conside ing ha , wi h a simple injec ion o he o ganic phase o e
he aqueous phase, he NCs pa icle size could be e icien ly educed,
we wan ed o know whe he his esul was composi ion-dependen .
To do so, we es ed di e en combina ions o componen s om a
panel o nanosys ems (Figu e 2b). Posi i e and nega i e nanoemul-
sions (NEs) and NCs we e p epa ed ei he by pou ing o by injec ing
he o ganic phase o e he aqueous phase (Figu e 2a), a a ixed con-
cen a ion o he componen s.
A conside able educ ion in he pa icle size was obse ed o all
he o mula ions when using he injec ion me hod (be ween 50 and
115 nm o size a ia ion; Figu e 2c). Fo leci hin/squalene NE (NE 4),
his dec ease was pa icula ly ob ious, wi h a d op in he pa icle size
om 171 3 o567 nm. This dec ease in size was also no iceable
when he pa icles we e analyzed by elec on mic oscopy (Figu e 2a).
Howe e , i is impo an o men ion ha he o mula ions ob ained
h ough injec ion ended o ha e sligh ly highe polydispe si y index
(PDI), especially o NEs, a ac ha e lec s he s abilizing p ope y o
he polyme shell.
CRECENTE-CAMPO AND ALONSO 41
3.1.2 |P epa a ion o NCs wi h pa icle size >400 nm
In he same way ha he e is a educ ion in he size o he NCs when
he concen a ions o hei componen s in he o ganic and he aque-
ous phases a e dec eased, when hei concen a ions a e highe he e
is a subs an ial inc ease in he NCs size. Howe e , wi h he s anda d
p epa a ion p o ocols, he e is a limi beyond which, agg ega ion
and/o ee oil a e obse ed. In ou hands, pa icle sizes highe han
400 nm we e di icul o achie e wi h he p e iously epo ed p oce-
du es. To ob ain highe pa icle sizes, we de eloped a new 2-s ep
me hod o he p epa a ion o NCs consis ing o CS/leci hin/ i amin
E. The me hod is desc ibed as ollows.
3.1.2.1 |Fo ma ion o an uns able nanoemulsion
A e he addi ion o a small olume o wa e o a concen a ed solu-
ion o leci hin and i amin E in e hanol, he spon aneous o ma ion o
a dynamic colloidal sys em was obse ed. The sys em e ol ed du ing
he i s 2 min gi ing ise o an inc ease in he pa icle size and, e en-
ually, o he o ma ion o mac oscopic oily d ople s, p obably due o
he concen a ion and he size o he o med nanod ople s.
3.1.2.2 |S abilizing he nanoemulsion
A e he addi ion o wa e o he o ganic phase and, p io o he
agg ega ion phase (wi hin he i s 2 min), he sys em could be s abi-
lized by adding a second, and la ge , olume o wa e con aining
CS. By adjus ing he elapsed ime be ween he addi ion o he ini ial
olume o wa e added o he e hanolic phase and he addi ion o he
polyme solu ion, i was possible o modula e he NCs pa icle size in
he 200–500 nm ange.
To e alua e he in luence o he ini ial olume o wa e added o
he o ganic phase, (s ep 1, Figu e 3a), he elapsed ime be ween he
ini ial addi ion o wa e and he addi ion o he CS solu ion was kep
cons an . The esul s showed ha when he ini ial olume was small
(0.1–0.2 mL) compa ed wi h he e hanol olume (0.5 mL), he oil
emained solubilized in he mix u e and only a e adding a second
and la ge olume o polyme aqueous solu ion (s eps 2–4), NCs wi h
a size in he 250–350 nm ange we e o med. When he ini ial olume
o wa e inc eased up o 0.3–0.5 mL, we de ec ed he o ma ion o
la ge nanod ople s, which a e he addi ion o he CS aqueous
solu ion p oduced s able NCs wi h a e age pa icle sizes be ween
400 and 500 nm. Finally, when he ini ial olume o wa e in s ep
1was≥0.6 mL, he size o he NCs was simila o hose ob ained wi h
he p e iously epo ed p epa a ion p ocedu es (a ound 350 nm). In
Figu e 3b, he pa icle sizes o he ob ained NCs by his p ocedu e a e
compa ed wi h he s anda d p o ocol o NCs p epa a ion, wi h only one
addi ion o wa e wi h he polyme dissol ed in i o e he o ganic phase.
On he o he hand, he elapsed ime be ween he wo addi ions
o wa e is also c ucial because o he dynamic na u e o he esul ing
nanoemulsion, as discussed be o e. Fo ha eason, ime is ano he o
he a iables ha can be used o modula e he inal pa icle size, being
a30–60 s ange app op ia e o ob ain pa icles sizes la ge han
300 nm (Figu e 3c).
3.2 |Laye -by-laye su ace modi ica ion o NCs
Besides hei pa icle size, he su ace cha ge and composi ion o he
nanosys ems a e also impo an pa ame e s ha in luence hei inal
a e in i o.
61,62
In his ega d, he laye -by-laye (LbL) echnique
allows he modi ica ion o he nanosys em’s su ace and he inco po-
a ion o new compounds in he o mula ion.
63–65
We p e iously
epo ed he in e es in combining he LbL echnique wi h he
sol en -displacemen me hod, o p oduce bilaye dex an sulpha e
(DS)-CS NCs o an igen p o ec ion and a con olled deli e y.
24
The
goal o he ollowing expe imen s was o assess he applicabili y o
he assembling p ocess o a a ie y o polyme s (bilaye NCs), and o
de e mine he maximum numbe o laye s ha can be assembled
a ound he oily co es (mul ilaye NCs).
3.2.1 |Bilaye NCs
CS/leci hin/ i amin E monolaye NCs, used as a model empla e, we e
incuba ed wi h di e en a ios o sul a ed and ca boxyla ed polya-
nions: hyalu ona e (HA), algina e (Alg), and chond oi in sul a e (ChS)
(Figu e 4a). The coa ing wi h a bilaye is conside ed e ec i e when an
in e sion in he ζ-po en ial occu s, which indica es ha he posi i e
cha ge o CS has been comple ely masked by he nega i e cha ges o
he polyanion. The mass/mass a io a which his in e sion happens
is, ul ima ely, de e mined by he ela i e cha ge o he second poly-
me . In he case o he es ed polyme s, he cha ges pe monome a
FIGURE 1 Response su ace indica ing he impac o di e en pa ame e s on he nanocapsules pa icle size. The olumes o bo h he o ganic
and aqueous phase, and he way he i s phase was added o e he second (by pou ing o injec ing h ough a needle) we e a ied. The pa icle
size o he esul ing nanocapsules was plo ed agains hese h ee pa ame e s (a). In (b), he in luence o he aqueous phase olume and he a e
o addi ion o e he pa icle size we e ep esen ed. The olume o o ganic phase was kep cons an a 5 mL in his case
42 CRECENTE-CAMPO AND ALONSO

pH = 7 a e: HA (0.5) < Alg (1) = ChS (1). These cha ges explain why
wi h a small amoun o ChS ( a io CS:ChS 1:0.25), an impo an in e -
sion in he ζ-po en ial was al eady obse ed (Figu e 4b), an in e sion
simila o he one ound wi h Alg ( a io CS:Alg 1:0.25) (Figu e 4c), and sig-
ni ican ly smalle compa ed wi h he needed amoun o HA ( a io CS:HA
1:1) (Figu e 4d). These esul s a e in ag eemen wi h hose p e iously
epo ed o he bilaye CS/DS whe e a a io CS:DS o 1:0.1 in e ed he
ζ-po en ial,
24
due o he high nega i e cha ge pe monome o DS (2.3).
3.2.2 |Mul ilaye NCs
Mul ilaye NCs can inc ease he o mula ion load, and modi y o delay
he elease o d ug h ough he mul iple polyme ic shells. Based on
he bilaye NCs desc ibed abo e, we ex ended his me hodology o
he enginee ing o mul ilaye NCs. San os e al. ollowed a simila
app oach using a washless LbL polyelec oly e assembly o encapsu-
la e low solubili y d ugs.
66
In hei case, he deposi ion o he poly-
me s was assis ed by sonica ion. This could be a p oblem when
wo king wi h labile biomolecules. The g oup o P o . Benoi , an expe
in lipid NCs,
67,68
de eloped mul ilaye CS/DS NCs (6 laye s) using he
phase in e sion me hod, which needed a pu i ica ion s ep by angen-
ial low il a ion be ween he deposi ion o each laye .
46
Ou goal
was o de e mine he maximum numbe o laye s ha could be buil
o e he oily co e, wi hou pu i ica ion s eps, and wi hou high-ene gy
inpu s ha could dena u e labile molecules.
In he p ocess o enginee ing a mul ilaye NC, he key pa ame e
o be conside ed was he mass a io be ween he polyme s wi h
100 – 200 nm
Sample Polyme Su ac an Co-su ac an Oil
NE 1 - Leci hin - Vi amin E
NE 2 - TPGS Sodium chola e Vi amin E
NE 3 - Tween® 80 - Vi amin E
NE 4 - Leci hin - Squalene
NE 5 - Leci hin - Miglyol®
812
NE 6 - Leci hin CTAB Vi amin E
NC 1 Algina e Leci hin CTAB Vi amin E
NC 2 Hyalu ona e Leci hin CTAB Vi amin E
NC 3 Chi osan Leci hin - Vi amin E
NC 4 Polya ginine Leci hin - Vi amin E
(b) (c)
(a) Pou ing Injec ion
i i ii ii
NE1
NE2
NE3
NE4
NE5
NE6
NC1
NC2
NC3
NC4
0
50
100
150
200
Sys em
Pa icle size (nm)
Pou ing Injec ion
**
** **
***
***
***
*** ***
** ***
40 – 100 nm
FIGURE 2 Reduc ion o he nanocapsules pa icle size ollowing he injec ion o he o ganic phase o e he aqueous phase. The injec ion o he
o ganic phase inside he aqueous phase in he p epa a ion o nanoemulsions (NEs) and nanocapsules (NCs) by he sol en -displacemen
echnique ema kably educed he pa icle size compa ed wi h jus pou ing one phase o e he o he . FESEM images, using STEM (i) and InLens
(ii) de ec o s, o he CS/leci hin/ i amin E NCs p epa ed by he wo me hods. Scale ba 200 nm. (a) Di e en combina ion o oils, su ac an s and
polyme s we e es ed o p oduce NEs and NCs upon pou ing o injec ing he o ganic phase o e he aqueous phase. (b) Di e ences in he pa icle
size o he NCs and NEs ob ained wi h his modi ica ion in he p epa a ion p ocedu e a e shown in C. ** and *** deno e signi ican di e ences
be ween samples (p< .01 and p< .001, espec i ely)
CRECENTE-CAMPO AND ALONSO 43
opposi e cha ges (CS and DS). This mass a io had o be empi ically
calcula ed o ensu e an e icien coa ing wi h he minimum amoun
possible o ee polyme .
24
We did his o a oid he p esence o any
soluble polyme in he colloidal suspension ha could con ibu e o
he o ma ion o undesi ed subpopula ions o polyme ic nanocom-
plexes, h ough he in e ac ion wi h he polyme added o build he
nex laye . O he impo an pa ame e o conside was he a io
be ween he olume o he NCs suspension and he polyme solu ion
added o o m he new laye (Suppo ing In o ma ion Figu e S2).
Using his echnique, up o 5 laye s o polyme s could be buil o e
he NE, wi hou he need o pu i ica ion s eps (Figu e 5a,c). The cho-
sen a io DS:CS was 0.5:1 o he i s wo laye s, o he hi d (CS),
ou h (DS), and i h (CS) laye s he a io polyme :CS- i s -laye was
1:1 wi h a inal a io CS
5 h
/DS
4 h
/CS
3 d
/DS
2nd
/CS
1s
1:1:1:0.5:1. An
in e sion o he ζ-po en ial was obse ed wi h each new polyme
laye added: om highly posi i e, when he CS was in he ex e nal
laye , o highly nega i e, when he DS was he ex e nal polyme
(Figu e 5d). Mo eo e , he addi ion o CS inc eased he pa icle size
when i coa ed he NE o NCs, as expec ed. On he con a y, DS
sligh ly educed he pa icle size when added o e CS NCs, p obably
because i s highly nega i e cha ge caused a con ac ion o he
polyme s.
3.3 |Scale-down o he NCs p epa a ion: A high-
h oughpu sc eening-adap able p ocedu e
The high- h oughpu sc eening (HTS), using obo ics and au oma ic
p ocesses, has allowed he p oduc ion and es ing o a huge numbe
o compounds in a e y sho pe iod o ime,
69–71
leading o unp ece-
den ed ad ances in he pha macology ield.
72
Apa om sa ing ime,
a HTS-adap able p ocedu e allows he p epa a ion o mul iple nano-
sys ems a once, wi h di e en composi ion o a io be ween compo-
nen s, hus mul iplying exponen ially he possibili ies o success when
sc eening nanosys ems.
In his s udy, a 96-mul iwell pla e was used o p epa e NCs in a
HTS-adap able p ocedu e, wi h a educed ba ch olume (300 μl). Fo
Uns able
nanoemulsion
S able
nanocapsules
Phase
sepa a ion
1
2
3
4
Wa e
>2min
Polyme
aqueous
solu ion
O ganic
phase
00.52 51030
0
200
400
600
800
1000
#
##
##
##
Time (min)
Pa icle size (nm)
(a) (b)
(c)
i ii
0 0.1 0.2 0.3 0.4 0.5 0.6
0
200
400
600
800
Ini ial wa e olume (mL)
Pa icle size (nm)
*
***
****
**
FIGURE 3 P epa a ion me hod o nanocapsules wi h pa icle sizes > 400 nm. Schema ic ep esen a ion o he p ocedu e o p epa e
nanocapsules wi h a pa icle size >400 nm. This me hod consis s o wo s eps: In a i s s ep, a dynamic nanoemulsion is o med by adding a small
olume o wa e o e an o ganic phase; in a second s ep, he p ima y nanoemulsion is dilu ed wi h a la ge olume o a polyme aqueous solu ion
o o m s able nanocapsules. FESEM images, using STEM (i) and InLens (ii) de ec o s, o he esul ing nanocapsules. Scale ba 200 nm (a).
In luence o he ini ial olume o aqueous phase in he inal pa icle size o he nanocapsules (b). E olu ion o he nanoemulsion pa icle size a e
he ini ial olume o wa e was added, showing i s dynamic na u e (c). # Indica es he p esence o la ge pa icles in some eplica es, ## indica es
he p esence o la ge pa icles in all eplica es
44 CRECENTE-CAMPO AND ALONSO
0.00 0.25 0.50 1.00 2.00
0
100
200
300
400
500
-80
-60
-40
-20
0
20
40
60
a io ChS:CS (w/w)
Pa icle size (nm)
Pa icle size
-po en ial
𝝵-po en ial (mV) 𝝵-po en ial (mV)
𝝵-po en ial (mV)
0.00 0.25 0.50 1.00 2.00
0
100
200
300
400
500
-80
-60
-40
-20
0
20
40
60
a io HA:CS (w/w)
Pa icle size (nm)
Pa icle size
-po en ial
Agg ega es
0.00 0.25 0.50 1.00 2.00
0
100
200
300
400
500
-80
-60
-40
-20
0
20
40
60
a io Alg:CS (w/w)
Pa iclesize(nm)
Pa icle size
-po en ial
Agg ega es
Agg ega es
(c)
(a) (b)
(d)
Hyalu onic acid Alginic acid
Chond oi in sulpha e
R
1
= SO
3
H;
R
2
,R
3
= H
FIGURE 4 Sc eening o he a io o posi i e: nega i e polyme used o p epa e bilaye nanocapsules. (a) S uc u e o he di e en
polysaccha ides used o p epa e bilaye nanocapsules o chi osan (CS) and sodium hyalu ona e (HA) (b), Sodium algina e (Alg) (c), and sodium
chond oi in sulpha e (ChS) (d) in hei acid o m. Do line indica es neu al ζ-po en ial
(a) (b)
(c) (d)
FIGURE 5 Sc eening o di e en chi osan: dex an sulpha e polyme a ios o s udy he o ma ion o mul ilaye nanocapsules. Di e en chi osan
(CS)/dex an sulpha e (DS) a ios we e used o o m he bilaye nanocapsules (NCs) (a). Fo he ollowing laye s, he mass a io e e ed o he
i s laye o CS was a ied o o m he hi d-laye and ou h-laye o NCs (b and c, espec i ely). The i h laye was achie ed di ec ly using a
a io 1:1 be ween he CS o he i s and i h laye s. Fo he es o he laye s he a io o elec ion is indica ed wi h he s iped ba . E olu ion o
he pa icle size and ζ-po en ial depending on he numbe o laye s (d)
CRECENTE-CAMPO AND ALONSO 45
his pu pose, a selec ion o 3 di e en oils, 4 su ac an s, 2 co-su ac-
an s, and 7 polyme s we e combined o p oduce 12 di e en NEs
and 84 di e en NCs in he same mul iwell pla e (Figu e 6). Such an
on-demand sc eening o o mula ion condi ions has ne e been
epo ed be o e. Taking in o conside a ion ha some o he es ed
compounds a e p esen in ma ke ed accines (such as i amin E, squa-
lene, and Tween 80),
73
and ha mos o he polyme s ha e shown
immunos imulan p ope ies when associa ed o an igens in a nano-
pa icula ed o m,
74
we en isage ha hese nano o mula ions migh
ha e a eal po en ial as nano accines once loaded wi h he equi ed
an igen.
Apa om showing he easibili y o minia u izing he NCs p epa-
a ion p ocedu e, and hus acili a ing a as sc eening o mul iple
nanosys ems a he same ime, his expe imen allowed us o d aw
some conclusions ha a e a he speci ic o he nanosys ems
desc ibed he e (Suppo ing In o ma ion Table S1). Fo example,
al hough mos o he nanosys ems had a pa icle size be ween
150 and 250 nm, squalene NEs/NCs ended o ha e la ge sizes and,
in some cases, a wide dis ibu ion (PDI > 0.3). The combina ion o
i amin E in he oily co e and poloxame 407 (P407) as a su ac an
ende ed he smalles nanosys ems. On he o he hand, NCs wi h ca -
boxyme hyl be a glucan (CM-β-glucan) as polyme ended o ha e
la ge sizes and highe PDI.
3.4 |Scale-up o he NCs p epa a ion
The s anda d p ocedu e o he p epa a ion o NCs by sol en dis-
placemen usually in ol es an o ganic phase, equen ly e hanol, a
hal he olume o he wa e phase;
39
howe e , ou esul s show ha
educing he pe cen age o e hanol om 33.33% o 4.76%, did no
a ec he s abili y o he NCs (Suppo ing In o ma ion Figu e S3).
Apa om minimizing/a oiding he use o sol en s, ano he challenge
he indus y aces when de eloping a nano o mula ion is he
(a)
(b)
Vi amin E Squalene Miglyol 812
TPGS T80 Lec P407 TPGS T80 Lec P407 TPGS T80 Lec P407
-
- - -- 0 - 0 -- - 0 0 -- 0
Polya ginine
++ ++ ++ ++ + 0 ++ + 0 0 ++ ++
Chi osan
+ + ++ + 0 0 ++ + + 0 ++ +
Hyalu ona e
-- -- -- --- -- -- -- -- -- ---
Dex an
sulpha e
- - -- --- -- -- -- -- -- -- --
Algina e
-- -- -- -- -- -- -- -- -- -- -- --
Polyglu ama e
-- -- -- - - - -- - - - -- --
CM-β-glucan
-- -- -- -- --- -- -- - - -- --
Pa icle size Ze a po en ial
< 150 nm ++ > +30 mV
150 – 200 nm + +10 – +30 mV
200 – 250 nm 0 +10 – -10 mV
250 – 300 nm - -10 – -30 mV
> 300 nm -- < -30 mV
-
Polya ginine
Chi osan
Hyalu ona e
Dex an sulpha e
Algina e
Polyglu ama e
CM-β-glucan
TPGS
T80
Lec
P407
TPGS
T80
Lec
P407
TPGS
T80
Lec
P407
Vi amin E Squalene Miglyol 812
CTAB
FIGURE 6 Minia u iza ion o he nanocapsules p epa a ion p ocedu e in an adap able-HTS me hod. Di e en combina ions o polyme s, oils,
and su ac an s we e used o p epa e a a ie y o nanocapsules in a 96-mul iwell pla e (a). A schema ic ep esen a ion o he pa icle size and
ζ-po en ial o he esul ing nanosys em (b). CM-β-glucan: Ca boxyme hyl-β-glucan, T80: Tween 80, Lec: Leci hin, P407: Poloxame 407, CTAB:
Ce yl ime hylammonium b omide
46 CRECENTE-CAMPO AND ALONSO