Self-assembled hyaluronan nanocapsules for the intracellular delivery of anticancer drugs

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Sel -assembled hyalu onan
nanocapsules o he in acellula
deli e y o an icance d ugs
Ana cade e1,2, Ana oli e a1,2, Magnus Bese 3, P adeep K. Dhal3, Lídia Gonçal es
4,
An ónio J. Almeida4, Guillaume Bas ia 5, Jean-Pie e Benoi 5, Ma ía de la uen e6,7,
Ma cos Ga cia- uen es
1,2, Ma ía José Alonso1,2 & Dolo es o es2
p epa a ion o sophis ica ed deli e y sys ems o nanomedicine applica ions gene ally in ol e mul i-
s ep p ocedu es using o ganic sol en s. In his s udy, we ha e de eloped a simple sel -assembling
p ocess o p epa e doce axel-loaded hyalu onic acid (HA) nanocapsules by using a sel -emulsi ica ion
p ocess wi hou he need o o ganic sol en s, hea o high shea o ces. These nanocapsules, which
comp ise an oily co e and a shell consis ing o an assembly o su ac an s and hyd ophobically modi ied
HA, ha e a mean size o 130 nm, a ze a po en ial o −20 mV, and exhibi high doce axel encapsula ion
e iciency. The nanocapsules exhibi ed an adequa e s abili y in plasma. Fu he mo e, in i o s udies
pe o med using A549 lung cance cells, showed e ec i e in acellula deli e y o doce axel. On he
o he hand, blank nanocapsules showed e y low cy o oxici y. O e all, hese esul s highligh he
po en ial o sel -emulsi ying HA nanocapsules o in acellula d ug deli e y.
Nanomedicine esea ch aims a de eloping he apeu ic agen s o add ess ch onic and se ious human diseases.
P og ess in nanomedicine esea ch o cance he apy has p o ided new oppo uni ies o he de elopmen o
mul i- unc ional nanoca ie s, wi h he po en ial o imp o e issue and o gan speci ic in acellula deli e y o
an icance d ugs wi h minimal o a ge oxici y1,2. Ve sa ili y o nanoma e ials enables one o design nano-
ca ie s o di e se physical and chemical p ope ies o achie e speci ic s uc u es and unc ions. Nanocapsules,
which comp ise an oily co e su ounded by a polyme shell, ha e gained special a en ion due o hei e sa ile
s uc u es and unable physicochemical p ope ies3,4. The oil co e has he abili y o e icien ly encapsula e hyd o-
phobic molecules, while he polyme ic shell endows he ca ie wi h desi able pha maceu ical cha ac e is ics,
such as d ug p o ec ion, ex ended s abili y and a ge ing5. The a ional choice o s a ing ma e ials is key o c ea e
nanocapsules wi h op imal physicochemical cha ac e is ics, low oxici y, and high loading capaci y o a ge and
kill cance cells6.
Hyalu onic acid (HA) is a unique and e sa ile anionic na u al polyme , which is ubiqui ous in na u e. I is
p oduced by i ually e e y issue in highe o ganisms and some bac e ia. High molecula weigh HA ha is ee
o con amina ing p o eins and nucleo ides is non-immunogenic. P esence o a la ge numbe o ca boxyl and
hyd oxyl g oups in HA allows acile chemical modi ica ion o conjuga e bioac i e agen s7,8. Fu he mo e, HA is
a known ligand o CD44 ecep o s ha a e o e exp essed in many umo ypes. These ea u es combined wi h i s
biodeg adabili y make HA an a ac i e building block o p epa e d ug loaded nanocapsules9.
P e iously, we ha e de eloped HA-based nanocapsules by using a sol en displacemen me hod whe e he
nega i ely cha ged HA shell was associa ed wi h a ca ionic su ac an by elec os a ic in e ac ions10. Chemically
modi ied HA ca ying hyd ophobic side chains could be an in e es ing al e na i e o p epa e nanocapsules.
Such a polyme is expec ed o sel -assemble wi hou he need o any ca ionic su ac an s, which could lead
o elimina ion o inhe en oxici y o ca ionic su ac an s11. Wi h espec o impa ing desi ed hyd ophobici y,
1Nanobio a G oup, IDIS, CIMUS, Uni e si y o San iago de Compos ela, San iago de Compos ela, Spain.
2Depa men o Pha maceu ics and Pha maceu ical Technology, School o Pha macy, Uni e si y o San iago de
Compos ela, San iago de Compos ela, Spain. 3Sano i-Global R&D, Sano i US, Wal ham, USA. 4Resea ch Ins i u e o
Medicines (iMed.ULisboa), Facul y o Pha macy, Uni e sidade de Lisboa, Lisbon, Po ugal. 5Mic o e Nanomedecines
T ansla ionnelles, MINT, Uni e si é Ange s, INSERM 1066, CNRS 6021, 4 ue La ey, Ange s, F ance. 6Nano-
Oncology Uni , T ansla ional Medical Oncology G oup, Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS),
SERGAS, San iago de Compos ela, Spain. 7Cance Ne wo k Resea ch (CIBERONC), Mad id, Spain. Co espondence
and eques s o ma e ials should be add essed o D.T. (email: [email p o ec ed])
Recei ed: 18 Ap il 2019
Accep ed: 22 July 2019
Published: xx xx xxxx
open
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HA de i a i es can be p epa ed wi h app op ia e deg ee o subs i u ion, wi hou a ec ing i s ecep o a ge ing
cha ac e is ics12. These amphiphilic HA de i a i es ha e al eady been used o he o ma ion o micelles wi h
he help o sonica ion13–15 o elec os a ic in e ac ions16 o he encapsula ion o hyd ophobic d ugs like doxo u-
bicin. Howe e , his is he i s epo on he use o hyd ophobically modi ied HA o p epa e nanocapsules by a
sel -emulsi ying p ocedu e. Since hese polyme ic nanocapsules can be p epa ed wi hou using o ganic sol en s,
hea o mechanical s i ing, his o e s an a ac i e me hod o inco po a e he mosensi i e molecules including
pep ides, p o eins, and an ibodies17.
Sel o spon aneous emulsi ica ion is a low ene gy me hod mos ly desc ibed o he p epa a ion o nano-
emulsions18–22. Using his p ocess, he o ma ion o nanosized d ople s is mainly dependen on he modula ion
o he in e acial phenomenon and he in insic physicochemical p ope ies o oils and su ac an s23. In he same
way as nanoemulsions, polyme ic nanocapsules can be p epa ed wi hou o ganic sol en s, hea o mechanical
s i ing, p o iding ad an ages om he manu ac u ing and scale-up s andpoin . Mo eo e , he de elopmen o
o mula ion echniques wi h less o ganic sol en s and lowe ene gy le els is expec ed o ha e a posi i e impac
in he en i onmen , as well as on he inal p oduc ion cos s24. In his manusc ip , we epo o he i s ime he
use o dodecyl side chain con aining HA as he p ecu so polyme o p epa e nanocapsules by a spon aneous
emulsi ica ion p ocess. Doce axel was success ully loaded in HA-based nanocapsules. Po en ial he apeu ic u ili y
o hese nanocapsules o u he imp o e in acellula deli e y o doce axel was e alua ed in i o by using A549
lung cance cells.
Resul s
Syn hesis and cha ac e iza ion o dodecylamide- unc ionalized sodium hyalu ona e.
Dodecylamide- unc ionalized sodium hyalu ona e (C12-HA) was syn hesized by eac ing e abu ylammonium
sal o HA wi h 1-aminododecane by using 2-b omo-1-e hyl py idinium e a luo obo a e as he amide coupling
eagen . The eac ion p ocess is shown in Fig.1.
The eac ion yield o each syn he ic s ep was be ween 50–70%. C12-HA was cha ac e ized by 1H NMR spec-
oscopy using a B uke A ance 400 MHz NMR spec ome e . A ep esen a i e NMR spec um, aken a 30 °C, is
shown in Fig.2. Analysis o he spec um con i ms he s uc u e and composi ion o he polyme . Fo example,
he peak a 1.93 ppm (a) co esponds o he me hyl p o ons o he ace amido moie y o HA. Fu he mo e, he
peaks a 1.22 ppm and 0.92 ppm co espond o he me hylene ((CH2)10) and e minal me hyl p o ons, espec i ely
o he dodecylamide side chain. The deg ee o subs i u ion (DS) o he dodecylamide g oup was de e mined om
he peak a ea a io o he me hyl g oups o he ace amide g oup o HA (a) and he me hyl g oup o dodecylamide
subs i uen (c) (Fig.2). The DS o e di e en lo s was ound o be in he ange o 2.5% o 5.0%. The syn hesis p o-
cess has been epea ed se e al imes and has been ound o be highly ep oducible.
P epa a ion o HA nanocapsules. In he i s s ep, he expe imen al condi ions equi ed o he o ma-
ion o a nanoemulsion (wi hou he HA shell) wi hou using o ganic sol en s, high empe a u e o shea o ces
we e in es iga ed. The componen s o p epa ing such nanoemulsions included Miglyol®812 and Tween®80 o
he oil phase and Solu ol®HS15 in he aqueous solu ion. Fo mula ion op imiza ion was ca ied ou by a ying
he ollowing componen s: (1) he concen a ion o Solu ol®HS15 solu ion, (2) he a io be ween Miglyol®812
and Tween®80 in he oil phase, and (3) he a io be ween he oil and he aqueous phases. The esul s (shown in
Table1) show ha he size o he nanoemulsions anged be ween 138–169 nm wi h a polydispe si y index o ≤0.2.
The concen a ion o Solu ol®HS15 in he aqueous phase did no signi ican ly change he globule size and he
polydispe si y index. The e o e, he smalles concen a ion (2.5 mg/mL) was ixed o subsequen s udies in ended
o analyze he e ec o Miglyol®812/Tween®80 (w/w) and he oil/aqueous phase a ios ( / ). The Miglyol®812/
Tween®80 (w/w) a io selec ed was 1:1 since i led o he smalles pa icle size (Table1). Subsequen ly, he oil/
aqueous phase a io was a ied om 1:1 o 1:8 ( / ) wi hou a ec ing he physicochemical p ope ies o he
nanoemulsions.
Based on he abo e indings, he ollowing condi ions we e used o he o mula ion o nanoemulsions: he oil
phase was composed o Miglyol®812/ Tween®80 in a a io 1:1 (w/w) and he aqueous phase o a Solu ol®HS15
solu ion o 2.5 mg/mL. The oil phase was pou ed in o he aqueous phase in a a io 1:8 ( / ) and s i ed a 900 pm
du ing 20 min.
Figu e 1. Reac ion scheme o he syn hesis o dodecylamide unc ionalized sodium hyalu ona e. (A) (i)
Dowex 50WX8-400 (ii) e abu ylammonium hyd oxide. (B) (i) 2-b omo-1-e hyl py idinium e a luo obo a e
(BEP), dodecylamine (ii) Dowex 50WX8-400 (iii) Sodium Hyd oxide.
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Fo he nanocapsules o ma ion, wo di e en app oaches we e employed depending on he ou e laye ma e-
ial. Fo unmodi ied HA NCs, he polyme was a ached by elec os a ic o ces, so he oily co e was p e iously
ca ionized by adding he ca ionic su ac an Ce yl ime hylammonium b omide (CTAB) o he oily phase. In he
case o nanocapsules wi h a C12-HA laye , we ook ad an age o he amphiphilic na u e o his ma e ial, which
enabled us o a oid he use o any ca ionic su ac an .
Ca ionic nanoemulsions, used as co es o HA NCs, we e ini ially p epa ed by adding di e en amoun s (0.05,
0.10 and 0.15 mg/mL) o CTAB o he oil phase. The ca ionic su ac an p omo ed an in e sion o he nega i ely
cha ged nanoemulsion o posi i ely cha ged one. Fu he mo e, inc easing he amoun o CTAB esul ed in a
highe ze a po en ial, wi hou in luencing he mean d ople size (See Supplemen a y TableS1). Since no u he
ze a po en ial inc ease was obse ed wi h addi ional CTAB in he o mula ion, he concen a ion o his su -
ac an was ixed a 0.15 mg/mL o p epa e NCs based on unmodi ied HA. The o ma ion o he HA shell a ound
he oily nano-co es by adding 0.25 mg/ml o HA o he wa e phase esul ed in a shi o he ze a po en ial om
+10 mV o −19 mV (Fig.3). When he C12-HA de i a i e was used o o m he nanocapsules, he physicochem-
ical p ope ies o he sys em did no change app eciably (Table2). Howe e , highe concen a ions o C12-HA
we e equi ed o change he ze a po en ial as compa ed o hose equi ed p e iously wi h unmodi ied HA. As
Figu e 2. Rep esen a i e
1H NMR (400 MHz) spec um o dodecylamide unc ionalized sodium hyalu ona e in D2O.
Fo mula ion a iables NEs
cha ac e iza ion*
Solu ol®HS15 conc. (mg/mL) Miglyol®812/Tween®80 a io (w/w) Oil/aq. phase a io ( / ) Size (nm)
2.5
1:1 1:2
138 ± 3
5 138 ± 2
15 149 ± 3
25 140 ± 1
2.5
1:1
1:2
138 ± 3
1.5 :1 147 ± 3
2 :1 164 ± 1
3.5 :1 159 ± 3
2.5 1:1
1:3 139 ± 2
1:4 144 ± 1
1:5 152 ± 3
1:8 138 ± 3
Table 1. E ec o a ia ion in expe imen al pa ame e s on he physicochemical p ope ies o he
nanoemulsions p epa ed by sel -emulsi ica ion. *Polydispe si y index was 0.2 o all o mula ions excep 3.5:1
Miglyol®812/Tween®80 a io which was 0.3.
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such, a concen a ion o 0.5 mg/mL o C12-HA was needed o gi e a nega i e cha ge a ound −20 mV o he
nanocapsules (Fig.3). De ails on he composi ion and cha ac e iza ion o bo h ypes o nanocapsules a e shown
in Table2.
Size dis ibu ion and TEM images (Fig.4) con i med he p oposed mo phology o HA NCs consis ing o a
monodispe se sys em composed o an oil co e su ounded by a polyme ic shell.
The s abili y o bo h HA-based nanocapsules was es ed unde s o age condi ions a 4 °C o 6 mon hs. Unde
hese condi ions, bo h o mula ions we e ound o be e y s able, wi hou signi ican changes in pa icle size,
polydispe si y index, o ze a po en ial o up o 6 mon hs (See Supplemen a y TableS2).
S abili y o HA nanocapsules in human plasma. Unmodi ied HA and C12-HA based NCs we e incu-
ba ed in human plasma a 37 °C. Bo h ypes o nanocapsules main ained hei sizes upon incuba ion o up o
24 h (Fig.5).
P epa a ion and cha ac e iza ion o doce axel-loaded HA nanocapsules. The solubili y o
doce axel (DCX) in Miglyol®812 was 2.0 ± 0.2 mg/mL a oom empe a u e. As shown in Table2, he encapsula-
ion e iciency was close o 90% and he p oduc ion yield ~90% (Table3). Impo an ly, such high encapsula ion
alues did no a ec he size and PDI o he nanocapsules.
In i o elease assays. The elease p o ile o DCX om he nanocapsules was assessed by a me hod based
on he d ug ans e o an oily compa men 25. DCX was eleased om HA NCs and C12 HA NCs ollowing a
biphasic p o ile, showing an ini ial bu s elease o 55% and 45%, espec i ely, ollowed by a sus ained elease o
24 h (Fig.6).
In i o cy o oxici y o emp y and DCX-loaded HA nanocapsules. Cy o oxici y o emp y and loaded
HA and C12-HA NCs was assessed agains A549 lung adenoca cinoma cells a e 72 h and 48 h o incuba ion,
Anionic NE
Ca ionic NE
HA NCs
C12-HA
NCs
-30
-20
-10
0
10
20
Ze a Po en ial (mV)
Figu e 3. Su ace cha ge o di e en nanoca ie s p epa ed by spon aneous emulsi ica ion. Shi in he ze a
po en ial o anionic o ca ionic nanoemulsions (NE) was achie ed by CTAB addi ion (0.15 mg/mL). The
addi ion o HA (0.25 mg/mL) o C12-HA (0.5 mg/mL) o he aqueous phase led o in e sion in he cha ge o
nega i e alues.
Composi ion & cha ac e iza ion
HA NCs C12-HA NCs
(mg/mL) (mg/mL)
Miglyol®812 59 59
Tween®80 58 58
Solu ol®HS15 2.5 2.5
CTAB 0.15 —
HA 0.25 —
C12-HA — 0.5
Size (nm) 137 ± 11 126 ± 5
PDI 0.2 0.2
Ze a Po en ial (mV) −19 ± 1 −20 ± 2
Table 2. Composi ion and physicochemical cha ac e iza ion o HA NCs and C12-HA NCs p epa ed by sel -
emulsi ica ion.
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espec i ely. A long incuba ion ime (up o 72 h) was chosen o ensu e ha cells would ha e su icien ime o
mul iply and o co ec ly measu e cell ac i i ies a ibu ed o cellula main enance and su i al26. Mo eo e ,
he o oxici y o he wo su ac an solu ions used o p epa e he nanocapsules was also assessed. Bo h ypes o
nanocapsules p ese ed he cell iabili y when es ed a concen a ions up o 350 μg/mL (Fig.7a). Fu he mo e,
Figu e 4. TEM images o HA nanocapsules (a,c), and he co esponding size dis ibu ion by DLS (b,d).
(a,b) HA NCs; (c,d) C12-HA NCs.
04812162024
0
50
100
150
200
Time (h)
Size (nm)
HA NCs
C12-HA
NCs
Figu e 5. E olu ion o he mean pa icle size o HA and C12-HA NCs incuba ed wi h human plasma, a 37 °C
o 24 h.
Fo mula ion Size (nm) PDI ZP (mV) EE% Yield %
HA NCs 140 ± 5 0.2 −18 ± 2 88 ± 9 93 ± 2
C12-HA NCs 145 ± 6 0.2 −20 ± 1 86 ± 3 88 ± 8
Table 3. Cha ac e iza ion o DCX-loaded HA NCs and C12-HA NCs a e pu i ica ion by SEC.

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C12-HA NCs showed no sign o cellula cy o oxici y e en when es ed a he highes concen a ion (1,000 µg/mL).
On he con a y, a signi ican cy o oxici y was obse ed o he ee su ac an mix u e wi h CTAB, whe e only
20% o cells su i ed a 350 μg/mL a e 72 h.
DCX-loaded HA-based NCs showed a dose dependen cy o oxici y agains A549 cells (Fig.7b). The e we e no
s a is ically signi ican di e ences be ween DCX-loaded nanocapsules o mula ions and ee DCX. The minimal
hal inhibi o y concen a ion (IC50) o bo h ypes o DCX-loaded nanocapsules was 10 μM DCX a 48 h. Blank
nanocapsules added a he same concen a ion as DCX-loaded NCs showed negligible oxici y.
In acellula up ake o HA-based nanocapsules. To e alua e he in acellula up ake o HA NCs and
C12-HA NCs, Nile ed was loaded in o bo h nanocapsules o mula ions and hei cellula up ake by CD44 o e -
exp essing A549 cells was obse ed by con ocal mic oscopy. As a con ol, cells we e exposed o a solu ion o he
ee luo opho e (same concen a ion as o nanocapsules), which was no in e nalized by he cells (Fig.8a). On
he o he hand, when cells we e exposed o ei he Nile ed-loaded HA o C12-HA nanocapsules, a high luo es-
cence ( ed colo ) was obse ed a he cy oplasmic le el. (Fig.8b,c).
Discussion
We ha e p e iously epo ed he abili y o unmodi ied HA-based nanocapsules o he in acellula deli e y o
DCX in cance cells10. These nanocapsules we e o med by he sol en -displacemen echnique and equi ed
he use o a ca ionic su ac an o acili a e he a achmen o he HA shell o he nanoemulsion co e. The objec-
i e o his wo k was o adap o he i s ime a spon aneous emulsi ica ion echnique o he p epa a ion o
HA nanocapsules by using amphiphilic HA. Using his echnology, we ha e success ully p epa ed nanocap-
sules o smalle size and monodispe se pa icle size. These nanocapsules exhibi ed s abili y in human plasma
036
0
25
50
75
100
12 18 24
Time (h)
%DCX
HA NCs
C12-HA NC
s
Figu e 6. Release p o ile o DCX om DCX-loaded HA NCs and DCX-loaded C12-HA NCs in PBS a 37 °C
o 24 h.
050 100
0
40
80
120
250 500 750 1000
Conc. nanoca ie (µg/mL)
Cell Viabili y (%)
HA NCs
C12-HA NCs
F ee
F ee T80+Solu ol+C
T80+Solu ol
TAB
0.0 2.5 5.0 7.5
0
40
80
120
25 50 75 100
Conc. DCX (nM)
CellViabili y (%)
HA NCs
C12-HA NCs
DCX-loaded HA NCs
DCX-loaded C12-HA NC
s
F ee DCX
a
b
Figu e 7. In i o cell oxici y o A549 cells a e (a) exposi ion o di e en concen a ions o emp y HA NCs,
C12-HA NCs and ee su ac an s mix u e o 72 h, and (b) ee DCX, DCX-loaded HA nanocapsules and
emp y HA nanocapsules o 48 h.
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and ha e shown high p opensi y o in acellula up ake. Fu he mo e, he use o an amphiphilic HA de i a i e
(dedecylamide- unc ionalized sodium hyalu ona e, C12-HA) enabled us o p epa e nanocapsules wi hou he
need o a ca ionic su ac an , he eby imp o ing he oxici y p o ile o he esul ing deli e y sys em.
By keeping he deg ee o subs i u ion in C12-HA in he ange o 2.5–5%, he amphiphilic HA was soluble
in wa e . Fu he mo e, such polyme s e ain he heological and biodeg adabili y p ope ies o na i e HA27.
Sel -emulsi ying HA nanocapsules we e p epa ed by spon aneous emulsi ica ion a e ca e ul sc eening o o -
mula ion condi ions. Due o he absence o o ganic sol en s, hea o high mechanical shea ing, he spon aneous
emulsi ica ion p ocess is mainly de e mined by he composi ion o he nanocapsules and no ably by he p esence
o speci ic su ac an s28. Being a medium chain iglyce ide, Miglyol® has be e nanoemulsi ying p ope y han
long chain iglyce ides29,30. In e ms o su ac an s, non-ionic su ac an s wi h a hyd ophilic/lipophilic balance
(HLB) in he ange o 12 o 18 we e ound o be p e e able. Such compounds ha e a ce ain hyd ophilici y and,
hence, can be easily dispe sed in o he aqueous phase31. Tween®80, due o i s low oxici y, is one o he widely
used su ac an s in sel -emulsi ying sys ems32. Solu ol® was included in he o mula ion o wo easons: (1) p es-
ence o a PEG chain in i s s uc u e may p o ide s abili y o he nanoca ie 33, and (2) i possesses he equi ed
high HLB (HLB = 14–16), along wi h an abili y o inhibi p-glycop o ein pumps. The inhibi ion o his memb ane
pump in cance cells would esul in highe in acellula d ug accumula ion29.
The o ma ion o HA NCs was achie ed by a aching he HA shell o he CTAB-s abilized oily co es h ough
elec os a ic complexa ion. Nanocapsules coa ed wi h C12-HA we e o med by di ec associa ion o he polyme
wi h oily co e wi hou he use o a ca ionic su ac an . The dodecyl chains o C12-HA acili a ed he deposi ion
o he polyme on o he nanoemulsion in e ace, which migh ha e been d i en by hyd ophobic in e ac ions.
The o ma ion o he C12-HA coa ing is expec ed o esul in an inc eased s abili y o he nanocapsule sys em30.
S abili y in human plasma is a c i ical pa ame e in designing nano o mula ions o in a enous adminis-
a ion and a ge ed deli e y. O en upon con ac wi h he blood s eam, nanoca ie s su e a disassembling o
agg ega ion p ocess, which may lead o hei un a o able biodis ibu ion34,35. The HA nanocapsules desc ibed
he e we e ound o be s able in plasma and no pa icle agg ega ion was obse ed.
The an icance agen DCX was selec ed as he d ug candida e o ou sys ems due o i s high hyd ophobici y
and ex ensi e his o y o clinical use36,37. We we e able o encapsula e DCX e icien ly wi hin he oil co e wi hou
changing he key physical pa ame e s. The high encapsula ion e iciency alues (>85%) ob ained o bo h ypes
o HA o mula ions we e no a ec ed ei he by he ype o polyme shell o he mechanism o nanocapsule o -
ma ion. This sugges s ha he a ini y o he d ug o he oil phase was he main d i ing o ce behind he loading
p ocess. DCX-loaded nanocapsules o mula ed wi h HA and C12-HA showed a biphasic d ug elease p o ile; an
ini ial bu s elease ollowed by a cons an elease (~70% elease in 24 h s.). This biphasic elease p o ile has been
ypically obse ed in o he HA-based nanocapsules, whe e he ini ial bu s elease has been a ibu ed o he
pa i ion o he d ug be ween he oil co e and he aqueous ex e nal medium38. I should be no ed ha he ini ial
bu s was ound o be educed in he case o C12-HA nanocapsules compa ed o unmodi ied HA nanocapsules
( om 55% o 45%). This beha io could be a ibu ed o he en anglemen o he hyd ophobic chain a he in e -
ace o he nanocapsules, he eby e a ding he di usion o he d ug om he oily co e o he ex e nal medium.
A e his ini ial bu s , he elease p ocess was simila o bo h o mula ions. Wi h a pka1 = 2.82 and pka2 = 3.42,
HA is nega i ely cha ged a pH abo e 4. Thus, i should main ain i s ionized o m in PBS medium (pH 7.4) and is
expec ed o s abilize he nanocapsules in he physiological en i onmen o blood. Fu he mo e, he hyd ophobic
chain in C12-HA may ende he nanocapsule a mo e compac a chi ec u e, hus p o ec ing DCX om being
easily eleased.
A dec ease in he cy o oxici y o C12-HA NCs was expec ed since he o mula ion o nanocapsules wi h he
hyd ophobically- unc ionalized HA elimina ed he need o ca ionic su ac an s. This was e iden in he di -
e ences in he cellula oxici ies o he wo nanocapsules a he highes es ed concen a ion (1 mg/mL). This
inding co ela es o he p esence o he ca ionic su ac an CTAB in high concen a ion, which is in ag eemen
wi h p e ious epo s39. In addi ion, he ma ked di e ence in iabili y be ween he HA-based nanocapsules and
he su ac an solu ion composed o Tween®80/Solu ol/CTAB a 350 μg/mL sugges s he bene icial e ec o HA
su ounding he su ac an laye as well as he co ec isola ion o he sys em om he ee su ac an s40. When
loaded wi h DCX, bo h nanocapsules we e aken up by A549 cells leading o inhibi ion o cance cell g ow h.
Figu e 8. In acellula up ake o Nile ed-loaded HA-based nanocapsules in A549 cells. (a) F ee Nile ed.
(b) Nile ed-loaded HA NCs. (c) Nile ed-loaded C12-HA NCs. Nuclei we e s ained wi h DAPI (blue). Nile Red
exhibi s ed luo escence.
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The high cellula up ake was obse ed o bo h HA and C12-HA nanocapsules loaded wi h he luo escen dye,
Nile ed. As can be seen by con ocal mic oscopy, s ong luo escen signals we e de ec ed in he cells cy oplasm
o bo h nanocapsules p o o ypes in compa ison o he ee luo opho e. Fu he mo e, he luo escen in ensi y
was simila o bo h ypes o Nile ed-loaded nanocapsules. This sugges s ha he unc ionaliza ion o HA wi h
he dodecylamide chain wi h a modes deg ee o subs i u ion does no appea o a ec HA’s binding a ini y o
CD44 ecep o s, o i s abili y o in e ac wi h he cell memb ane. I migh be a ibu ed o he o ien a ion o he
lipophilic chain owa ds he oily phase and he hyd ophilic anionic g oups o HA chain owa ds he ex e nal
aqueous phase41.
In summa y, by ca e ully designing and syn hesizing an amphiphilic HA de i a i e, we ha e success ully
applied a sol en - ee sel -emulsi ica ion me hodology o p epa e a new class o nanocapsules consis ing o an
oily co e and a shell made up o hyd ophobically modi ied HA. The esul ing nanocapsules we e able o encapsu-
la e hyd ophobic d ugs such as doce axel qui e e icien ly and showed con ol o e he elease o he d ug. These
nanocapsules exhibi ed low cy o oxici y and demons a ed o be e icien ly aken up by cance cells o media e a
he apeu ic e ec .
Me hods
Reagen s. Sodium hyalu ona e (Mw = 200 KDa) was p o ided by Sano i Genzyme, USA. Cap ylic/cap ic
iglyce ide (Miglyol®812) was a kind gi om C eme , Ge many. Polyoxye hylene so bi an monoolea e
(Tween®80), hexadecyl ime hylammonium b omide (CTAB), Nile Red, DAPI and plasma ( om human) we e
pu chased om Sigma-Ald ich, Spain. Mac ogol 15 hyd oxys ea a e ( o me adename Solu ol®HS15, cu en ly
designa ed Kollipho ° HS15) was acqui ed om BASF, Ge many. Dulbecco’s Modi ied Eagles Medium (DMEM)
was pu chased om The mo Fishe Scien i ic, Spain. All o he chemicals used we e o eagen g ade.
Syn hesis o dodecylamide- unc ionalized sodium hyalu ona e (C12-HA). An aqueous solu ion
(concen a ion 10 mg/mL) o 200 mg o sodium hyalu ona e was ea ed wi h 5 mL o Dowex 50WX8-400 esin
(1.7 milliequi alen s/mL, H+ o m; eshly washed wi h wa e /me hanol/wa e ). A e emo ing he esin by
il a ion, he esul ing polyme solu ion was ea ed wi h 40% (w/w) aqueous e abu ylammonium hyd oxide
solu ion un il he pH o he solu ion became 12.0. The en i e p ocess o esin ea men and e abu ylammonium
hyd oxide solu ion ea men was epea ed wice. The inal pH o he polyme solu ion was adjus ed o 7.5–8.0
by bubbling wi h CO2 ollowed by bubbling wi h N2. The polyme solu ion was subsequen ly concen a ed by
angen ial low using a 30 KDa cu -o Pellicon XL Biomax il e casse e (EMD Millipo e). The concen a e was
lyophilized o d yness.
To he e abu ylammonium hyalu ona e (400 mg) we e added 45 mL o DMF and 4 mL o monome hyl o -
mamide. To he esul ing polyme solu ion was added 8.8 mg o 2-b omo-1-e hyl py idinium e a luo obo a e
dissol ed in 1 mL DMF. A e s i ing o 1 h, a solu ion con aining 12 mg o 1-aminododecane and 1.50 mL o
ie hylamine in 1 mL DMF was added o he eac ion mix u e. The mix u e was s i ed a oom empe a u e o
48 h . Subsequen ly he eac ion mix u e was added d opwise o 150 mL o ace one/ e ahyd o-2-me hyl u an
(1:1) solu ion. The p ecipi a e was collec ed, dissol ed in 50 mL o deionized wa e , lyophilized o d yness and
edissol ed in 50 mL o deionized wa e .
The abo e solu ion was ea ed wi h 5 mL o Dowex 50WX8-400 esin and s i ed o 10 min. The esin was
il e ed o and washed wi h deionized wa e . The il a e was ea ed wi h 1 M NaOH un il he pH was 12.0. The
p ocedu e was epea ed wo mo e imes and he inal pH was hen adjus ed o 7.5–8.0 by i s bubbling CO2 ol-
lowed by bubbling wi h N2. The solu ion was subsequen ly concen a ed ia angen ial low using a 30 KDa cu -o
Pellicon XL Biomax il e casse e and he concen a e was lyophilized. The p oduc (C12-HA) was cha ac e ized
by 1H-NMR spec oscopy o con i m i s s uc u e and deg ee o subs i u ion (See Supplemen a y Me hods).
De elopmen o he sel -emulsi ica ion me hod – p ima y emulsions. The sel -emulsi ica ion
me hod was ini ially op imized o he p epa a ion o nanoemulsions, which was subsequen ly adap ed o p e-
pa e HA-based nanocapsules.
Oil in wa e (o/w) nanoemulsions we e p epa ed wi hou o ganic sol en s and hea , by using a single-s ep
spon aneous emulsi ica ion p ocess. B ie ly, an oil phase (con aining Miglyol®812 and Tween®80) was added o
an aqueous phase (composed o wa e and Solu ol®HS15). Miglyol®812 and Tween®80 we e i s mixed oge he
and he mix u e was subsequen ly pou ed in o he aqueous phase and s i ed a 900 pm o 20 min. The op imi-
za ion o he nanoemulsion composi ion was by a ying he a ious componen s in he ollowing manne :
(a) E ec o Solu ol®HS15 concen a ion in he aqueous phase
An oil phase composed o Miglyol®812 and Tween®80 (1:1 a io w/w) was added unde magne ic s i ing
o an aqueous phase (oil/aqueous phase a io 1:2 / ) con aining di e en concen a ions (2.5, 5, 15 and
25 mg/mL) o Solu ol®HS15.
(b) In luence o Miglyol®812/Tween®80 a io
An oily phase composed o Miglyol®812 and Tween®80 a a ios (1:1, 1.5:1, 2:1, and 3.5:1 w/w) was p e-
pa ed and pou ed in o an aqueous Solu ol®HS15 solu ion (oil/aqueous phase a io 1:2 / ). The concen a-
ion o Solu ol®HS15 was kep ei he a 2.5 o 25 mg/mL.
(c) In luence o oil/aqueous phase a io
The oily phase, composed o Miglyol®812/Tween®80 (1:1 a io w/w) was added o he Solu ol®HS15 solu ion
a he concen a ion o 2.5 mg/mL, while he oil/aqueous phase a ios we e a ied be ween 1:2 and 1:30 ( / ).
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P epa a ion and op imiza ion o HA nanocapsules. Using he op imized sel -emulsi ica ion p ocess
desc ibed abo e, wo ypes o HA-based nanocapsules we e p epa ed by using aqueous solu ions o sodium hya-
lu ona e (HA) o dodecylamide- unc ionalized HA (C12-HA). These we e called HA nanocapsules (HA NCs)
and C12-HA nanocapsules (C12-HA NCs), espec i ely. To p epa e HA NCs, he ca ionic su ac an CTAB was
dissol ed in he oil phase a di e en concen a ions (0.05, 0.10 and 0.15 mg/mL). Fo bo h p o o ypes, he hyalu-
onan (HA o C12-HA) solu ions we e used h ee di e en concen a ions (0.25, 0.5 and 1 mg/mL).
P epa a ion o doce axel-loaded HA nanocapsules. DCX was solubilized in Miglyol®812 and i s sol-
ubili y de e mined ollowing he p ocedu e o Saliou e al., wi h sligh modi ica ions42. DCX-loaded nanocapsules
we e p epa ed as desc ibed be o e by eplacing Miglyol®812 wi h he DCX-Miglyol®812 solu ion. Loaded DCX
was sepa a ed om he ee d ug by size exclusion ch oma og aphy (SEC). DCX was quan i ied by HPLC ollow-
ing he me hod o Ri e a-Rod iguez e al.43. Yield and encapsula ion e iciency (%) was calcula ed as desc ibed in
Supplemen a y in o ma ion (See Supplemen a y Me hods).
P epa a ion o luo escen dye loaded HA nanocapsules. Nile ed-loaded HA and C12-HA NCs we e
p epa ed as desc ibed be o e and he luo escen p obe was inco po a ed in o he oil co e. Encapsula ed Nile ed
was sepa a ed om he ee by SEC ollowing he de ined p o ocol.
Cha ac e iza ion o nanocapsules. Cha ac e iza ion o HA-based NCs was ca ied ou by measu -
ing hei mean pa icle size, polydispe si y index (PDI) and ze a po en ial (ZP) using dynamic ligh sca e ing
(DLS) (Ze asize Nano-ZS, Mal e n Ins umen s). Mo phological analysis was ca ied ou ansmission elec on
mic oscopy (TEM).
Assessmen o s abili y o nanocapsules in human plasma. HA NCs and C12-HA NCs we e dilu ed
1:10 ( / ) in human plasma o a pe iod o 24 h, a 37 °C. A p ede e mined ime in e als, samples we e aken and
pa icle sizes measu ed by he me hod desc ibed abo e.
Measu emen o physical s abili y o nanocapsules unde s o age condi ions. Fo he assessmen
o he long- e m s abili y o nanocapsules, samples we e kep undilu ed a 4 °C and s o ed o up o 6 mon hs. Size
and PDI we e e alua ed as desc ibed be o e.
In i o elease assays. In i o elease (IVR) assays we e assessed using a d ug ans e me hod adap ed
om Bas ia e al.25. This me hod was op imized o he IVR p o ile o DCX om sel -emulsi ying HA-based
nanocapsules unde sink condi ions. (See Supplemen a y Me hods).
In i o cy o oxici y assays. The cell iabili y Alama ®Blue was u ilized o assess he cy o oxici y o bo h
emp y and DCX-loaded HA-based NCs44. Se ial dilu ions o emp y nanocapsules we e added o A549 lung ade-
noca cinoma cells and incuba ed o 72 h. Simila ly, cells we e exposed o se ial dilu ions o ee DCX, emp y, and
DCX-loaded HA-based NCs (DCX concen a ions o 0.625, 1.25, 2.5, 5, 10 and 100 nM) o 48 h. A e incuba-
ion, samples we e wi hd awn and eplaced by esh medium con aining 5 mM Alama Blue®. Fluo escence was
measu ed a 530 and 590 nm (exci a ion and emission, espec i ely) using a mic opla e eade (Fluos a Omega,
BMG Lab ech, Ge many). The ela i e cell iabili y (%) compa ed o con ol cells was calcula ed as he pe cen age
o he luo escence o he samples di ided by he con ol (See Supplemen a y Me hods).
Cell up ake assay. Cellula up ake o Nile ed-loaded HA NCs was s udied in A549 cells. 60,000 cells/well
we e seeded in a co e glass and incuba ed wi h an app op ia e olume o he o mula ion equi alen o 50 ng o
luo opho e. A e dilu ing in DMEM, he suspension media was le o 4 h . A he end o his ime, he cells we e
ixed, s ained wi h DAPI, and we e isualized by con ocal mic oscopy (Leica, TCS SP5).
S a is ical analysis. Samples we e p epa ed a leas in iplica e and da a p esen ed as he mean ± s anda d
de ia ion (SD). Fo in i o cell assays, esul s a e shown as he mean ± SD o he da a ob ained in wo sepa a e
expe imen s wi h 3 eplica es in each expe imen (n = 6). S a is ical e alua ion o da a was pe o med using he
one-way analysis o a iance (ANOVA). Tukey–K ame mul iple compa ison es (G aphPad PRISM 5 so wa e,
La Jolla, CA, USA) was used o compa e he signi icance o he di e ence be ween he g oups, and a p < 0.05 was
accep ed as signi ican .
Da a A ailabili y
All da a gene a ed and analyzed du ing his s udy a e included in his published a icle and in he Supplemen a y
In o ma ion sec ion.
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