Mannose-modified hyaluronic acid nanocapsules for the targeting of tumor-associated macrophages

Vol.:(0123456789)
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D ug Deli e y and T ansla ional Resea ch
h ps://doi.o g/10.1007/s13346-022-01265-9
ORIGINAL ARTICLE
Mannose‑modi ied hyalu onic acid nanocapsules o  he a ge ing
o  umo ‑associa ed mac ophages
IagoFe nández‑Ma iño1,2,3· Clémen An ay4 · JoseC ecen e‑Campo1,2,3 · Akihi oMaeda4 ·
AldoUmma ino4 · Ca menTeijei o‑Valiño5 · Da ioBlanco‑Ma inez1,2,3· F ancisMpambani6· Lau encePoul6·
JulieDe allie e6· Ma hieuGe main6· JuanCo ea7· Ma cosFe nandez‑Villama in7· PaolaAlla ena4 ·
Edua doFe nandez‑Megia7 · Ma íaJoséAlonso1,2,3 · Fe nandoTo esAndón1,4
Accep ed: 16 No embe 2022
© The Au ho (s) 2022
Abs ac
Tumo -associa ed mac ophages (TAMs), a class o immune cells ha play a key ole in umo immunosupp ession, a e
ecognized as impo an a ge s o imp o e cance p ognosis and ea men . Consequen ly, he enginee ing o d ug deli e y
nanoca ie s ha can each TAMs has acqui ed special ele ance. This wo k desc ibes he de elopmen and biological e alua-
ion o a panel o hyalu onic acid (HA) nanocapsules (NCs), wi h di e en composi ions and p epa ed by di e en echniques,
designed o a ge mac ophages. The esul s showed ha plain HA NCs did no signi ican ly in luence he pola iza ion o
M0 and M2-like mac ophages owa ds an M1-like p o-in lamma o y pheno ype; howe e , he chemical unc ionaliza ion o
HA wi h mannose (HA-Man) led o a signi ican inc ease o NCs up ake by M2 mac ophages in i o and o an imp o ed
biodis ibu ion in a MN/MNCA1 ib osa coma mouse model wi h high in il a ion o TAMs. These unc ionalized HA-Man
NCs showed a highe accumula ion in he umo compa ed o non-modi ied HA NCs. Finally, he p e-adminis a ion o he
liposomal li e occupying agen Nanop ime ™ u he inc eased he accumula ion o he HA-Man NCs in he umo . This
wo k highligh s he p omise shown by he HA-Man NCs o a ge TAMs and hus p o ides new op ions o he de elopmen
o nanomedicine and immuno he apy-based cance ea men s.
Keywo ds Cance · Hyalu onic acid· Mannose· Polyme ic nanocapsules· Nanop ime · Tumo -associa ed mac ophages
Abb e ia ions
BSA Bo ine se um albumin
DLS Dynamic ligh sca e ing
FACS Fluo escence-ac i a ed cell so ing
FBS Fe al bo ine se um
HA Hyalu onic acid
HA-50 Hyalu onic acid o 50kDa
HA-330 Hyalu onic acid o 330kDa
HA-1500 Hyalu onic acid o 1500kDa
HA NCs Nanocapsules wi h a hyalu onic acid poly-
me ic coa ing
HA SD-NCs Nanocapsules p epa ed by a sol en -
displacemen echnique
Iago Fe nández-Ma iño, Clémen An ay, and Jose C ecen e-Campo
con ibu ed equally o his wo k.
Edua do Fe nandez-Megia, Ma ía José Alonso, and Fe nando
To es Andón sha ed senio au ho ship.
* Fe nando To es Andón
e nando. o [email p o ec ed]
1 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
2 Depa men o Pha macology, Pha macy andPha maceu ical
Technology, Campus Vida, Uni e sidade de San iago de
Compos ela, San iagodeCompos ela15782, Spain
3 Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS),
San iagodeCompos ela15706, Spain
4 Labo a o y o Cellula Immunology, IRCCS Humani as
Resea ch Hospi al, Rozzano-Milan20072, I aly
5 Nanomag Labo a o y, Applied Physics Depa men ,
Campus Vida, Uni e sidade de San iago de Compos ela,
San iagodeCompos ela15782, Spain
6 Cu adigm 60 ue de Wa ignies, Pa is75012, F ance
7 Depa amen o de Química O gánica, Cen o Singula de
In es igación en Química Biolóxica e Ma e iais Molecula es
(CIQUS), Uni e sidade de San iago de Compos ela, Jena o
de la Fuen e s/n, San iagodeCompos ela15782, Spain
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HA SE-NCs Nanocapsules p epa ed by a sel -
emulsi ying echnique
HA-Man Mannose- unc ionalized hyalu onic acid
HDMD Human monocy e-de i ed mac ophages
IC-50 Hal -maximal inhibi o y concen a ion
IL In e leukin
IFN-γ In e e on gamma
LPS Lipopolysaccha ide
Man Mannose
MHC-II Majo his ocompa ibili y complex class II
NCs Nanocapsules
NTA Nanopa icle acking analysis
PDI Polydispe si y index
hM-CSF Recombinan human monocy e colony-
s imula ing ac o
SE-NEs Nanoemulsion p epa ed by sel -emulsi ying
echnique
STEM Scanning ansmission elec on mic oscopy
TAMs Tumo -associa ed mac ophages
TNF-α Tumo nec osis ac o α
In oduc ion
Tumo -associa ed mac ophages (TAMs) play a majo ole in
umo p og ession [1]. They exhibi a ch onic-in lamma o y
pheno ype (M2-like) ha p omo es immunosupp ession
in he umo mic oen i onmen h ough he p oduc ion o
ch onic in lamma o y cy okines, in e leukins, p os aglandins,
and ans o ming g ow h ac o s [2, 3]. TAMs also modula e
o he aspec s o umo p og ession, such as angiogenesis, cell
p oli e a ion, ib osis, and me as asis [4]. The e o e, hese
immunosupp essi e cells a e a ele an a ge o cance
p ognosis and ea men [1, 4].
In pa allel, nano echnological app oaches s and ou o
hei e sa ili y o design a la ge numbe o nanos uc u es
adap ed o di e en he apeu ic objec i es, including TAMs
a ge ing [5, 6]. In his con ex , lipid nanopa icles [7, 8],
liposomes [9–12], and polyme ic nanopa icles [13, 14],
among o he nanoca ie s, ha e been designed o each
TAMs o p ognosis pu poses o as he apies o kill hem,
impai hei p o umo al unc ions, o ep og am hem owa ds
M1-like mac ophages wi h an i umo al ac i i y [6, 15].
Hyalu onic acid (HA) is an endogenous glycosaminogly-
can, in ol ed in he a chi ec u al s uc u e o issues, which
has been used as a polyme o design TAM- a ge ed nanoca -
ie s [16–18]. HA is ecognized by se e al ecep o s, among
hem he CD44 ecep o , which is widesp ead in cance ,
endo helial, and immune cells [19, 20]. Se e al s udies ha e
demons a ed ha he HA molecula weigh , HA/ ecep o
a io, and/o ecep o iso o ms could limi i s a ge ing capac-
i y [21]. Wi h he aim o exploi ing he passi e a ge ing
capaci y o nanos uc u es, ou esea ch g oup has de eloped
di e en ypes o HA-based nanoca ie s [22–26], pa icu-
la ly nanopa icles (NPs) and nanocapsules (NCs). NCs a e
e sa ile sys ems ha ha e he abili y o ha bo in hei oily
co e di e en ypes o d ugs, such as small hyd ophobic
molecules, bu also la ge mac omolecules, ei he in hei
ou e polyme ic laye o in hei in e phase. P e iously, we
ha e dedica ed in ensi e e o s o design HA NCs capable o
deli e ing an icance d ugs o he umo mic oen i onmen
[27]. Mo e p ecisely, we ha e de eloped doce axel-loaded
HA NCs ha show an impo an an i umo al e icacy in a
lung cance mu ine model [28]. We ha e also in es ed sig-
ni ican e o in adap ing HA-based nano echnologies o he
deli e y o polynucleo ides [29, 30], pep ides [31], p o eins
[23, 32], and monoclonal an ibodies [33], in he con ex o
cance . Ne e heless, he HA a ge ing capaci y has shown
mixed esul s, p obably due o he abo emen ioned wide-
sp ead p esence o he CD44 ecep o in di e en ypes o
cells. Thus, we ha e decided o explo e he unc ionaliza-
ion o HA wi h ac i e a ge ing ligands. Recen ly, we ha e
demons a ed ha HA unc ionalized wi h he umo homing
pep ide Lyp-1 inc eases subs an ially he accumula ion o
HA NCs in a lung cance model [28]. Following a simila
app oach, in his pape , we hypo hesize ha he unc ionali-
za ion o HA wi h ligands o ecep o s p esen on he su ace
o TAMs should imp o e hei in i o pe o mance.
The monosaccha ide mannose (Man) is one o he mos
well-known ligands used o he ac i e a ge ing o immune
cells. Man is in ol ed in he ecogni ion o endogenous and
pa hogenic molecules h ough he CD206 ecep o , exp essed
by dend i ic cells and mac ophages [34]. In e es ingly, he
CD206 ecep o is also a pheno ypic ma ke o e exp essed
by M2-like (i.e., TAMs) e sus M1-like mac ophages in solid
umo s [35]. In acco dance, mannosyla ed nanoca ie s ha e
been used o he p e e en ial a ge ing o TAMs [36–41].
Howe e , e y limi ed knowledge is s ill a ailable abou he
combina ion o HA and Man in a nanos uc u e o TAMs
a ge ing, which is he scope o ou cu en wo k.
He e, we desc ibe he de elopmen and e alua ion o a
panel o HA NCs designed o a ge TAMs. Di e en p epa-
a ion echniques and composi ions, including HA o di e -
en molecula weigh s (MWs) and Man- unc ionalized hya-
lu onic acid (HA-Man), led o a a ie y o p o o ypes which
we e ully cha ac e ized in e ms o hei physicochemical
p ope ies. P ima y human mac ophages we e exposed o
hese HA NCs o es hei immuno oxic ac i i y and in e -
naliza ion. Finally, in o de o unde s and hei abili y o
each mac ophages in solid umo s, he biodis ibu ion and
umo a ge ing abili y o HA-Man NCs we e analyzed, wi h
and wi hou he p e-adminis a ion o Nanop ime ™ (which
dec ease li e clea ance o NCs) in a ully immunocom-
pe en MN/MCA1 ib osa coma mu ine model wi h a high
in il a ion o TAMs.
D ug Deli e y and T ansla ional Resea ch
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Ma e ials andme hods
Ma e ials
Fo he syn hesis o HA‑Man All chemicals we e pu chased
om Sigma-Ald ich and we e used wi hou u he pu i i-
ca ion. Hyalu onic acid was pu chased om Li eco e Bio-
medical (sodium hyalu ona e, lo numbe 024168, speci-
ica ion numbe LDP-9800042; Mw 57kDa by MALLS).
2,3,4,6-Te a-O-ace yl-α-D-mannopy anosyl ichlo oace imi-
da e and 2-[2-(2-azidoe hoxy)e hoxy]e hanol we e p epa ed
ollowing p e iously epo ed p ocedu es [42–44]. All sol-
en s we e o HPLC g ade, pu chased om Scha lab and
Fishe . E 3N was d ied unde 4Å molecula sie es. CH2Cl2
was d ied using a SPS800 sol en pu i ica ion sys em om
MBRAUN. H2O o Milli-Q g ade was ob ained om a Milli-
po e wa e pu i ica ion sys em. Ambe li e IR-120 was sequen-
ially washed wi h H2O, MeOH, and CH2Cl2 be o e use.
Fo he p epa a ion o he NCs Cap ylic-cap ic iglyce -
ides (Miglyol® 812) we e ob ained om IOI Oleo GmbH
(Hambu g, Ge many). Ce imonium b omide, DL-α-
ocophe ol (Calbiochem®), and polyso ba e 80 (Tween®
80) we e ob ained om Me ck KGaA (Mad id, Spain).
Benze honium chlo ide was pu chased om Spec um
Chemical M g. Co p. (New B unswick, USA). Polye hyl-
ene glycol (15)-hyd oxys ea a e (Kollipho ® HS 15) was
pu chased om BASF SE (Ludwigsha en, Ge many).
DL-α- ocophe ol-TPGS was bough o An a es Heal h P od-
uc s Inc. (Jonesbo ough, USA). Leci hin-soya (Epiku on®
145V) was bough o Ca gill Inc. (Minne onka, USA). Fi y
and 1500kDa HA we e ecei ed om Li eco e Biomedi-
cal Inc. (Chaska, USA). In o al, 330kDa HA was ecei ed
om LEHVOSS I alia S l. (O iggio, I aly). DiD′; DiIC18(5)
(1,1′-dioc adecyl-3,3,3′,3′- e ame hylindodica bocyanine,
4-chlo obenzenesul ona e sal ) and DiR′; DiIC18(7)
(1,1′-dioac adecyl-3,3,3′,3′- e ame hylindo ica bocyanine
iodide) we e p o ided by The moFishe Scien i ic Inc. (Mad id,
Spain). Nanop ime ™ echnology was p o ided by Cu adigm
SAS (Pa is, F ance).
Syn hesis, isola ion, andcha ac e iza ion o HA‑Man
A solu ion o DMTMM (55mg, 0.199mmol) and 3
(7.45mg, 0.024mmol) in 1mM phospha e bu e (pH 6.5,
0.4mL) was added o a solu ion o HA (40mg, 0.10mmol)
in 1mM phospha e bu e (pH 6.5, 0.9mL). The eac ion
mix u e was s i ed a 70°C o 2h and hen was ul a il-
e ed (YM5; 4 × 30mL sa NaHCO3, 3 × 30mL H2O) and
lyophilized o a o d HA-Man (37.5mg, DS 15.6%; coupling
yield 65%; mass eco e y 85%). 1H NMR (500MHz, D il e
120ms, D2O) δ: 4.90 (s, 0.16H), 4.54 (b s, 1H), 4.45 (b s,
1H), 4.05–3.25 (m, 12.81H), 2.02 (s, 3H). Fo mo e de ails
abou he syn hesis o new compounds, see he supplemen-
a y ma e ials sec ion.
Column ch oma og aphy
Au oma ed column ch oma og aphy was pe o med on
a MPLC Teledyne ISCO CombiFlash RF 200 psi wi h
RediSep R columns e illed wi h silica 40–63µm ( om
VWR Chemicals) o neu al alumina oxide 60 mesh ( om
Fishe Scien i ic). Samples we e adso bed on o silica o neu-
al alumina and loaded in o solid ca idges.
NMR spec oscopy
NMR spec a we e eco ded on a Va ian Me cu y 300MHz
o Va ian Inno a 500MHz spec ome e s. Chemical shi s
a e epo ed in ppm (δ uni s) down ield om in e nal e a-
me hylsilane (CDCl3), in e nal 3-( ime hylsilyl)p opionic-
2,2,3,3-d4 acid sodium sal (D2O), o he esidual HOD peak
(D2O). De e mina ion o he subs i u ion deg ee o HA-Man
was done by ela i e in eg a ion be ween he 3H o he
N-ace yl g oup o HA (2.02ppm) and he anome ic p o on
o he Man pendan s (4.90ppm) in he 1H NMR spec um o
he polyme (D2O) eco ded wi h a 1H-di usion il e (s imu-
la ed Echo-LED pulse sequence wi h bipola PFG g adien s,
elaxa ion delay (d1) was se o 15s, and di usion delay (Δ)
was se o 120ms). Mes ReNo a 14.2 so wa e (Mes elab
Resea ch) was used o spec a p ocessing.
In a ed spec oscopy
FT-IR spec a we e eco ded on a Pe kinElme spec um wo
spec opho ome e equipped wi h an ATR accesso y.
Mass spec ome y (MS)
Mass spec a we e eco de on a B uke Mic o o spec om-
e e coupled o a HPLC Agilen 1100 using a mosphe ic-
p essu e chemical ioniza ion (APCI) o elec osp ay ioniza-
ion (ESI). Samples we e injec ed ia low injec ion analysis
(FIA) using a MeOH/aqueous solu ion o o mic acid 0.1%
1:1, low 0.2mL/min.
Ul a il a ion
Ul a il a ion was pe o med on Millipo e Amicon s i ed
cells wi h Amicon YM5 egene a ed cellulose memb anes
unde a 5 psi N2 p essu e.
D ug Deli e y and T ansla ional Resea ch
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HA NCs p epa a ion
HA SE‑NCs
The p epa a ion p o ocol o HA SE-NCs was adap ed om
a p e iously desc ibed p o ocol [33, 45]. Mo e p ecisely,
0.875mL o a 2.75mg/mL benze honium chlo ide solu-
ion in polyso ba e 80 was added o 1mL o cap ylic-cap ic
iglyce ides o ob ain a s ock solu ion o he oily phase.
To ob ain he wa e phase, 0.5mL o an aqueous solu-
ion o polye hylene glycol (15)-hyd oxys ea a e (7.5mg/
mL); 0.25mL o an aqueous solu ion o HA o 50, 330, o
1500kDa (1.5mg/mL); and 0.575mL o ul apu e wa e
we e mixed in o a beake . Then, 0.175mL o he oily phase
was quickly added o e he wa e phase (1.325mL) unde
magne ic s i ing. Finally, he esul ing HA SE-NCs we e
isola ed using Cen ipu eP10 size exclusion columns (EMP
Bio ech GmbH.; Be lin, Ge many) o a inal 1.5mL olume.
HA SD‑NCs
The p epa a ion p o ocol o HA SD-NCs has been p e iously
desc ibed [46]. Mo e p ecisely, o ob ain he oily phase, he
ollowing solu ions in e hanol we e mixed in a mic o ube:
0.2mL o DL-α- ocophe ol (67.5mg/mL), 0.2mL o DL-α-
ocophe ol-TPGS (20mg/mL), and 0.1mL o benze honium
chlo ide (5mg/mL). Then, 1mL o an aqueous solu ion
o HA o 50kDa (2mg/mL) and ul apu e wa e (0.5mL)
we e added in o a beake o ob ain he wa e phase. The oily
phase was added quickly o e he wa e phase unde mag-
ne ic s i ing o conclude he emulsi ica ion phase. Las ly,
he o ganic sol en s we e emo ed by e apo a ion unde he
ume hood, and he inal olume was adjus ed o 2mL wi h
ul apu e wa e .
HA SD‑NCs/HA‑Man SD‑NCs loaded wi h luo escen dyes
o up ake andbiodis ibu ion s udies
The p epa a ion p o ocols o HA SD-NCs and HA-Man SD-
NCs ha e been p e iously desc ibed [28]. Mo e p ecisely,
0.2mL o an 18.75mg/mL e hanolic solu ion o leci hin-soya,
0.05mL o a 15mg/mL e hanolic solu ion o ce imonium
b omide cap ylic-cap ic iglyce ides (0.015mL), and ace one
(4.75mL) we e added in o a es ube o ob ain he oily phase.
The wa e phase, o med by 10mL o non-modi ied o man-
nose-modi ied HA o 50kDa (0.25mg/mL), was placed in o
a beake . The oily phase was added o e he wa e phase d op
by d op unde magne ic s i ing. Finally, he o ganic sol en s
we e emo ed wi h a o a o y e apo a o and he inal olume
adjus ed wi h ul apu e wa e up o 5mL. When needed, HA
SD-NCs and HA-Man SD-NCs we e labelled wi h DiD o
DiR, inco po a ing he luo opho es in o he oily phase wi h a
inal concen a ion o 25µg/mL.
Physicochemical cha ac e iza ion o HA NCs
DLS
HA NCs we e cha ac e ized by dynamic ligh sca e ing (DLS)
using a Ze asize Nano ZS ZEN 3600 equipmen (Mal e n
Panaly ical L d.; Mal e n, UK). P e ious o hei cha ac e i-
za ion, HA NCs we e dilu ed in ul apu e wa e (HA SE-NCs
1:100; HA/HA-Man SD-NCs 1:20). The measu emen pa am-
e e s we e he same o all he samples (lase inciden angle
173°, 25°C, 6 uns, 12s pe un, 3 measu emen s).
NTA
Selec ed HA NCs we e cha ac e ized by nanopa icle ack-
ing analysis (NTA) using a NanoSigh NS300 equipmen
(Mal e n Panaly ical L d.; Mal e n, UK). P e ious o hei
cha ac e iza ion, HA NCs we e dilu ed in ul apu e wa e
(HA SE-NCs 1:100; HA/HA-Man SD-NCs-TG 1:20). The
measu emen pa ame e s we e he same o all he sam-
ples (lase wa eleng h 488nm, 25°C, 5 cap u es, 60s pe
cap u e).
STEM
Scanning ansmission elec on mic oscopy (STEM) images
we e aken om selec ed HA NCs. Fi s ly, HA NCs we e
dilu ed in ul apu e wa e (HA SE-NCs 1:10,000; HA/HA-
Man SD-NCs-TG 1:2,000) and mixed wi h he same olume
o a 2% (w/ ) phospho ungs ic acid solu ion. Then, samples
we e loca ed on coppe g ids wi h ca bon ilms and washed
wi h 1mL o ul apu e wa e . Once he g ids we e d ied,
hey we e obse ed in he mic oscope (FESEM; ZEISS,
ULTRA Plus, Ge many).
Colloidal s abili y o HA NCs
Colloidal s abili y a s o age condi ions
The main physicochemical p ope ies o he HA NCs we e
e alua ed as desc ibed in he “DLS” sec ion o 14days du -
ing he s o age o he HA NCs a 4°C.
Colloidal s abili y in ele an biological mediums
The main physicochemical p ope ies o he HA NCs, incu-
ba ed wi h RPMI/10% e al bo ine se um (FBS) o 24h,
we e e alua ed as desc ibed in he “DLS” sec ion.
D ug Deli e y and T ansla ional Resea ch
1 3
Endo oxin assessmen
The endo oxin con en o all he NCs was e alua ed wi h
PYROGENT™ Plus Gel Clo LAL and Kine ic-QCL®
Kine ic Ch omogenic LAL (Lonza G oup L d.; Basel, Swi -
ze land). Endo oxin con en o all samples was below he
limi o 0.125 EU/mL.
Isola ion anddi e en ia ion o HMDM
Human monocy e-de i ed mac ophages (HMDM) we e iso-
la ed om heal hy blood dono s, as p e iously desc ibed
[47]. HMDM we e ob ained by wo-s ep g adien cen i u-
ga ion using lymphocy e-H cell sepa a ion media (Me ck
KGaA.; Milan I aly) and Pe coll™ (The moFishe Scien i ic
Ins; Milan I aly). M0 mac ophages we e di e en ia ed by
cul u ing 1 × 106 HMDM in RPMI/5% FBS wi h 25.00ng/
mL o ecombinan human monocy e colony-s imula ing
ac o -1 ( hM-CSF-1) (Pep oTech Inc.; London, UK). M1
mac ophages we e di e en ia ed by s imula ing M0 in
RPMI/5% FBS wi h 100ng/ml o lipopolysaccha ide (LPS)
and 50ng/mL o in e e on-gamma (IFN-γ) (Pep oTech Inc.;
London, UK). Las ly, M2 mac ophages we e di e en ia ed
by s imula ing M0 in RPMI/5% FBS wi h 20ng/mL o in e -
leukin-4 (Pep oTech Inc.; London, UK).
Cy o oxici y assays
HMDM we e seeded in a 96-wells pla e a a densi y o
1 × 105 cells/wells. Cells we e incuba ed wi h HA NCs a he
indica ed concen a ions and imes in RPMI/10% FBS. Cell
iabili y was e alua ed wi h Ala ma Blue™ cell iabili y
eagen (The moFishe Scien i ic Inc.; Milan, I aly). Non-
ea ed cells we e used as 100% cell iabili y con ol.
Exp ession o mac ophages pheno ypical ma ke s
byFACS andcy okine sec e ion byELISA
M0 and M2 mac ophages we e seeded in a low-a achmen
24-wells pla e a a densi y o 1 × 106 cells/well and incuba ed
in RPMI/10% FBS wi h 100µg/mL o HA NCs o 48h.
T ea men wi h IFN-γ + LPS was used as induced posi i e
con ol. Cells we e esuspended in luo escence-ac i a ed
cell so ing (FACS) bu e (PBS/1% bo ine se um albumin
(BSA)) and labelled wi h Pe pCP™-Cy5.5 mouse an i-
human majo his ocompa ibili y complex class II (MHC-
II) and FITC mouse an i-human CD206 (BD Biosciences;
San Jose, USA). Finally, cells we e analyzed wi h he FACS
equipmen BD Can o™ (BD Biosciences; San Jose, USA).
The sec e ion le els o he umo al nec osis ac o α (TNF-α)
we e measu ed wi h an ELISA ki ollowing he ins uc ion
p o ided by he manu ac u ing company (R&D Sys ems
Inc.; Minneapolis, USA).
Up ake andin e naliza ion assays inmac ophages
M1 and M2 mac ophages we e seeded in a low-a achmen
24-wells pla e a a densi y o 1 × 106 cell/well. Cells we e
incuba ed wi h 0.50mL o RPMI/10% FBS con aining
0.5mg/mL o DiD-labelled HA SD-NCs o DiD-labelled
HA-Man SD-NCs o 1h.
Flow cy ome y
M1 and M2 mac ophages we e ixed o 20min a 4°C wi h
ixa i e solu ion (PBS/4% pa a o maldehyde), cen i uga ed,
and esuspended in FACS bu e . Cells we e analyzed wi h
FACS equipmen BD Can o™ (wa eleng hs: λexc 640nm,
λems 670nm).
Con ocal mic oscope
M1 and M2 mac ophages we e seeded in a 24-wells pla e
wi h co e slips (14nm). Cells we e labelled wi h DAPI and
ixed o 20min a 4°C wi h ixa i e solu ion (PBS/4% pa a-
o maldehyde). Glass co e slips we e eco e ed, moun ed,
and analyzed wi h a con ocal mic oscope Leica TCS SP8
SMD (Leica Came a Ag.; We zla , Ge many) (wa eleng hs:
λexc 640nm, λems 670nm).
Fib osa coma mice model
Female 6-week-old C57BL/6 mice we e used as animal
model. An o ho opic immunocompe en ib osa coma
model was gene a ed as p e iously desc ibed [48]. 1 × 105
MN/MCA1 cells we e injec ed in o he caudal high mus-
cle. P ocedu es in ol ing animals we e conduc ed ollowing
I alian (4D.L.N.116, G.U., supplemen 40,18–2-1992) and
Eu opean (Di ec i e 2010/63/EU) no ma i e. E o s we e
made o minimize he numbe o animals used and hei
su e ing.
Biodis ibu ion imaging
Mice we e sha ed o a oid he in e e ences o he u and
ed wi h an imaging die . 0.10mL o DiR-labelled HA SD-
NCs and DiR-labelled HA-Man SD-NCs we e adminis-
e ed by in a enous injec ion. Nanop ime ™ was injec ed
a 360mg/kg; 10min be o e NCs adminis a ion ollow-
ing manu ac u e p o ocol (Cu adigm SAS; Pa is, F ance).
Ke amine and xylazine a a concen a ion o 100mg/kg and
10mg/kg we e used o anes hesia. Finally, mice we e sac-
i iced wi h CO2.

D ug Deli e y and T ansla ional Resea ch
1 3
Analysis o IVIS images
Imaging analysis was pe o med wi h IVIS equipmen
Lumina III Sys em (Pe kinElme Inc; Wal ham, USA). Mice
we e inse ed in a supine posi ion in o he equipmen . Tis-
sues o in e es we e emo ed and analyzed ex i o. IVIS
equipmen was used wi h he same con igu a ion o all he
mice and issues (wa eleng hs: λexc 750nm, λems 780nm;
binning 4 o 8; /s op 2).
FACS analysis o excised umo s
Tumo s we e collec ed and p epa ed o FACS analysis.
Cells we e s ained wi h LIVE/DEAD® Fixable Aqua Dead
Cell S ain (In i ogen; 1:1000 in PBS -/-) o 30min a
oom empe a u e (RT) and hen s ained wi h he mix o
an ibodies (CD45-Pe CP—Clone 30-F11 (BD Biosciences);
Cd11b-APC eFluo 780—Clone M1/70 (eBiosciences);
F4/80-PE—Clone Cl:A3-1 (BioRad)), in FACS bu e o
30min a 4°C. Cells we e washed wi h FACS bu e and
ixed wi h FACS ix Bu e (1% PFA PBS) o 20min a
4°C. Cells we e analyzed on a FACS Can o™ II analyze
and da a gene a ed by FloJow so wa e (BD Biosciences).
S a is ical analysis
S a is ical analysis was pe o med wi h G aphPad P ism
so wa e. S a is ical compa isons we e done using wo-
way ANOVA wi h Tu key’s mul iple compa ison es and
one-way wi h Tu key’s mul iple compa ison es . Da a we e
exp essed as he mean ± s anda d de ia ion. P alues 0.05
o less we e conside ed s a is ically signi ican . *(p < 0.05);
**(p < 0.01); ***(p < 0.001); ****(p < 0.0001).
Resul s anddiscussion
Design, p epa a ion, andcha ac e iza ion o HA NCs
As a i s s ep owa ds he de elopmen o HA NCs
unc ionalized wi h mannose o he a ge ing o TAMs,
we ha e p epa ed a se ies o NCs wi h HA o di e en
molecula weigh s (MWs), using di e en p epa a ion
echniques and composi ions. In o de o do his, we ha e
buil on he expe ience o ou esea ch g oup in he design
and syn hesis o polyme ic nanocapsules (NCs) by sol en
displacemen (SD) o sel -emulsi ica ion (SE) echniques
[49–52]. As a con inua ion o ou p e ious wo k, he e we
s udy he ele ance o he o mula ion pa ame e s and
composi ion o HA NCs, including hei unc ionaliza-
ion wi h mannose, o hei in e ac ion wi h mac ophages
in he umo mic oen i onmen . We de eloped a panel o
HA NCs by he SE echnique using HA o di e en MWs
(50, 330, 1500kDa) (Fig.1). The polyme ic coa ing and
an oily co e o cap ylic-cap ic iglyce ides o hese p o-
o ypes (HA-50/HA-330/HA-1500 SE-NCs) we e simila
o he ones ha ha e al eady shown good in acellula
mAb deli e y [33, 45]. The coun e pa nanoemulsions,
wi hou he polyme ic coa ing (SE NEs), we e included
as con ol. Mo eo e , based on ou p e ious wo k using
doce axel-loaded nanosys ems [28], NCs p epa ed by he
SD echnique wi h a 50kDa HA polyme ic coa ing and an
oily co e o DL-α- ocophe ol (HA SD-NCs) [46] we e also
included in he panel (TableS1).
All HA NCs showed simila physicochemical p ope -
ies, wi h a pa icle size be ween 120 and 150nm (PDI
0.2–0.3) and Z-po en ial anging om sligh ly nega i ely
cha ged, o HA SE-NCs, o highly nega i e, o HA SD-
NCs (Table1) [53]. A deepe analysis o selec ed p o o-
ypes om he SE NCs g oup was pe o med, because due
o hei mo e ecen de elopmen by ou g oup, hey lack
he SD NCs exhaus i e cha ac e iza ion. Thus, HA-50 SE-
NCs and hei co esponding NE we e cha ac e ized wi h
o hogonal echniques, such as NTA and STEM, ollow-
ing he EU-NCL ecommenda ions [54]. These echniques
added ele an in o ma ion abou pa icle size, size dis i-
bu ion, and pa icle shape [55]. The HA-50 SE-NCs and
he co esponding nanoemulsions (SE-NEs) showed simila
pa icle size and dis ibu ion (Table2). Mo eo e , STEM
images showed pa icles wi h a sphe ical shape a ound
100nm (Fig.2). Finally, he whole panel o HA NCs we e
Fig. 1 Illus a ion o he hyalu onic acid nanocapsules (HA NCs)
Table 1 DLS cha ac e iza ion o HA NCs (n = 3)
Nanoca ie Pa icle size (nm) PDI Ze a po en ial
SE-NEs 139 ± 13 0.25 + 3 ± 1mV
HA-50 SE-NCs 130 ± 4 0.25 − 13 ± 3mV
HA-330 SE-NCs 133 ± 3 0.25 − 18 ± 2mV
HA-1500 SE-NCs 123 ± 5 0.25 − 19 ± 3mV
HA SD-NCs 151 ± 8 0.15 − 36 ± 3mV
D ug Deli e y and T ansla ional Resea ch
1 3
cha ac e ized ega ding hei s abili y in s o age condi ions
and ele an biological medium. Ou esul s show ha he
HA NCs we e s able a 4°C o a leas 15days and in
RPMI/10% FBS a 37°C o , a leas , 24h (Fig.S1).
Toxicological e alua ion o HA NCs
To e alua e he oxici y o he nanos uc u es in i o, human
monocy e–de i ed mac ophages (HMDMs) we e exposed
o di e en concen a ions o HA NCs o 24 and 48h. All
nanosys ems showed good ole abili y up o a concen a ion
o 100µg/mL. Only he NCs p epa ed by he sol en displace-
men echnique (HA SD-NCs) a a concen a ion o 100µg/
mL showed a mino oxici y, p esen ing a hal -maximal
inhibi o y concen a ion (IC-50) o 209.7µg/mL a 24h and
116.3µg/mL a 48h (Fig.3A) (TableS2). The highe oxici y
o hese HA SD-NCs is likely ela ed o he con en o ca ionic
su ac an in hei s uc u e [56]. All HA SE-NCs, p epa ed
wi h HA o di e en MWs, showed simila oxici y a 24h,
wi h IC-50 a ound 1000µg/mL (Fig.3A) (TableS2). O e all,
hese esul s demons a e he good biocompa ibili y o all he
de eloped HA NCs and allow o he selec ion o non- oxic
doses o u he in i o expe imen s as desc ibed below.
E ec o HA NCs on hepheno ype andpola iza ion
o mac ophages
Wi h ega d o he immunomodula o y p ope ies o HA,
o he s ha e sugges ed a p o-in lamma o y ac i i y o low
molecula weigh HA (< 150kDa) e sus an i-in lamma o y
p ope ies o high molecula weigh HA (> 1000kDa) [57].
Thus, we decided o es he immunomodula o y p ope ies
o ou HA NCs se ies (using HA o 50, 330, and 1500kDa).
Fo his, p ima y human mac ophages, M0 (non-pola ized)
and M2 (IL-4 ea ed) HMDMs, we e exposed o 48h o
he HA NCs. Incuba ion o HMDMs wi h IFN-γ + LPS was
used as posi i e con ol o M1-like pola iza ion (an i umo
mac ophages). The CD206 (mannose ecep o ) was quan i-
ied as M2-like ma ke , while, as indica ion o M1-like pola -
iza ion, he amoun o MHC-II ecep o on he su ace o
mac ophages was measu ed by FACS. The esul s in Fig.3B
indica e ha none o he HA SE-NCs induced signi ican
a ia ions in he exp ession le els o he CD206 and MHC-
II ecep o s in M0- o M2- ea ed mac ophages. Howe e ,
M0 and M2 mac ophages exposed o HA SD-NCs showed a
signi ican educ ion in he amoun o CD206 ecep o , which
could be ela ed o he oxici y o his o mula ion a 48h
(Fig.3A, B). O e all, no signi ican a ia ions we e obse ed
in he MHC-II ecep o o any o he HA NCs in M0- o
M2-exposed mac ophages, indica ing no abili y o induce
M1-like pola iza ion (as obse ed o he posi i e con ols).
To alida e hese esul s, he sec e ion o TNF-α by M0 and
M2 mac ophages incuba ed o 48h wi h 100µg/mL o he
di e en HA NCs se ies was e alua ed by ELISA. None o
he o mula ions es ed was able o induce he sec e ion o
his p o-in lamma o y cy okine (Fig.S2). These esul s a e in
ag eemen wi h p e ious esea ch by Miz ahi e al. [58] ha
showed ha mul ilamella esicles coa ed wi h polyme ic
HA o di e en MWs did no a ec he di e en ia ion o
RAW 264.7 mac ophages. As a whole, ou expe imen s using
human p ima y mac ophages indica e ha nei he he MW
o HA no he di e en su ac an s used in he SE o he SD
echniques (see de ailed composi ion in TableS1) signi i-
can ly a ec ed he pola iza ion o mac ophages.
Table 2 NTA cha ac e iza ion
o selec ed HA NCs (n = 3)
D10/50/90 dis ibu ion o he 10%/50%/90% o he pa icle popula ion, DLS dynamic ligh sca e ing, HA
SD-NCs nanocapsules p epa ed by sol en displacemen wi h a 50-kDa HA polyme ic coa ing and an oily
co e o DL-α- ocophe ol, HA SE-NCs nanocapsules p epa ed by sel -emulsi ying wi h a HA polyme ic
coa ing, NTA nanopa icle acking analysis, PDI polydispe si y index, SE-NEs nanoemulsion p epa ed by
sel -emulsi ying wi hou polyme ic shell, σ s anda d de ia ion
Nanoca ie Mean (nm) σ (nm) D10 (nm) D50 (nm) D90 (nm) Pa icles pe mL
SE-NEs 115 ± 6 44 74 104 168 9·1012 ± 2·1012
HA-50 SE-NCs 108 ± 5 37 72 99 144 6·1012 ± 2·1012
Fig. 2 STEM images o A SE-NEs; B HA-50 SE-NCs. EHT = 20.00kV; WD = 2.4mm Mag = 200.00K X. EHT, elec on high ension; Mag,
magni ica ion; STEM, scanning ansmission elec on mic oscopy; WD, wo k dis ance
D ug Deli e y and T ansla ional Resea ch
1 3
Syn hesis o mannosyla ed hyalu onic acid
(HA‑Man) andde elopmen o HA‑Man NCs
To imp o e he abili y o HA NCs o a ge mac ophages in
he TME, we implemen ed he unc ionaliza ion o he NCs
wi h mannose, as a a ge ing ligand. I is well-known ha he
mannose ecep o (CD206) is o e exp essed in TAMs (wi h
an M2 pheno ype) e sus in M1 and M0 (non-pola ized)
mac ophages [2]. Thus, we syn he ized a new HA polyme
unc ionalized wi h mannose (Man) esidues, as ligands o
be exposed on he su ace o he NCs o he ac i e a ge ing
o he CD206 ecep o s on he su ace o TAMs.
The mannosyla ed HA (HA-Man) was p oduced by amide
coupling be ween he ca boxylic acid a he D-glucu onic acid
monome s o HA and an amina ed mannose de i a i e (3),
Fig. 3 A HA NCs oxici y
p o ile owa ds HMDM a 24
and 48h; Alama Blue™ cell
iabili y assay. B Quan i ica-
ion o CD206 (M2 pheno ypic
ma ke ) and MHC-II (M1 phe-
no ypic ma ke ) on he su ace
o M0 and M2 mac ophages
a e 48-h incuba ion wi h HA
NC, measu ed by FACS. M1
mac ophages ( ea ed wi h
IFN-γ + LPS) we e used as
posi i e con ol. S a is ical
compa ison was pe o med
using one-way and wo-way
ANOVA wi h Tukey’s mul iple
compa ison es . *(p < 0.05);
**(p < 0.01); ***(p < 0.001);
****(p < 0.0001) espec o
con ol g oup. RFI, ela i e
luo escence in ensi y
Fig. 4 Syn hesis o mannosyla ed hyalu onic acid (HA-Man)
D ug Deli e y and T ansla ional Resea ch
1 3
p o ided wi h a lexible and hyd ophilic linke o ensu e an
e ec i e ecep o -media ed ecogni ion by he co esponding
cells (Fig.4). The amine 3 was p epa ed in h ee s eps om an
ace yla ed mannose ichlo oace imida e p ecu so [43, 59].
Fi s , a BF3-ca alyzed glycosyla ion wi h 2-[2-(2-azidoe hoxy)
e hoxy]e hanol a o ded he α-mannose de i a i e 1 in 72%
yield. The α con igu a ion a he anome ic posi ion o 1 was
con i med by 1H NMR acco ding o he obse ed J = 1.7Hz
coupling cons an (see supplemen a y in o ma ion). Dep o-
ec ion o he ace yl g oups (KOH, MeOH, 95%), ollowed
wi h azide educ ion by hyd ogena ion (1a m, Pd/C, 97%)
a o ded 3 in excellen yield. Amide coupling be ween HA
(50kDa) and amine 3 was pe o med unde sligh ly acidic
aqueous condi ions in he p esence o 4-(4,6-dime hoxy-l,3,5-
iazin-2-yl)-4-me hylmo pholinium chlo ide (DMTMM)
[42], a wa e -s able iazine-based condensing eagen . In e -
es ingly, he yield using DMTMM was supe io o he one
ob ained when using EDC/NHS [60]. The esul ing HA-Man
was ob ained wi h an 85% mass eco e y a e pu i ica ion by
ul a il a ion. A deg ee o subs i u ion (DS) o 16% was de e -
mined by ela i e in eg a ion in he 1H NMR spec um o he
polyme (D2O) be ween he N-ace yl p o ons o he HA chain
(2.02ppm) and he anome ic p o on o he mannose pendan s
(4.90ppm) (see supplemen a y in o ma ion). Conside ing ha
wo monosaccha ides comp ise he epea ing uni o HA, his
esul s in a deg ee o subs i u ion o 8% o ou polyme on a
monosaccha ide basis.
Using he HA-Man polyme , we p epa ed NCs by he SD
echnique (HA-Man SD-NCs), and hei physicochemical and
biological p ope ies we e compa ed wi h hose o he co e-
sponding non-modi ied NCs (HA SD-NCs). I is wo h men-
ioning ha ou esea ch g oup has p e iously demons a ed
he e sa ili y o simila HA SD-NCs o be unc ionalized
wi h pep ide a ge ing ligands, such as -LyP-1. The -LyP-1-
HA-NCs showed a signi ican imp o emen in hei biodis i-
bu ion p o ile, d ug accumula ion in he umo (when loaded
wi h doce axel), and a be e an i umo al e icacy in an A549
non-small lung cance animal model [28]. In he cu en
wo k, HA SD-NCs and HA-Man SD-NCs showed simila
physicochemical p ope ies, despi e he Man unc ionaliza-
ion (Table3). HA SD-NCs and HA-Man SD-NCs we e also
cha ac e ized wi h NTA and STEM. Mino modi ica ions in
he p epa a ion o HA SD-NCs o in i o expe imen s we e
pe o med a his s ep, as desc ibed in he me hodology “HA
SD-NCs/HA-Man SD-NCs loaded wi h luo escen dyes o
up ake and biodis ibu ion s udies” sec ion, and a comp e-
hensi e cha ac e iza ion o bo h o mula ions was comple ed
(ou esul s ound no signi ican di e ences be ween hem).
HA-Man SD-NCs p esen ed a 20nm la ge mean diame e
and a mo e he e ogeneous popula ion compa ed o HA SD-
NCs (Table4). The mean pa icle size de e mined by NTA
was smalle han he one obse ed wi h DLS o bo h p o o-
ypes. In bo h cases, STEM images showed sphe ical pa i-
cles wi h a size o abou 100nm (Fig.5).
In e naliza ion o mannosyla ed
andnon‑mannosyla ed HA NCs bymac ophages
To e alua e hei up ake by mac ophages, bo h HA NCs and
HA-Man NCs desc ibed in he p e ious sec ion we e now
labelled wi h DiD, and in e naliza ion assays we e e alua ed
by FACS and con ocal mic oscopy. Fi s , p ima y human
M1 (an i umo al) and M2 (p o umo al) mac ophages we e
cul u ed in i o, and he p esence o he CD206 ecep o
on he su ace o he cells o each pola iza ion s a us was
measu ed. As expec ed, he highes alue was obse ed o
he M2 mac ophages, mimicking TAMs [35] (Fig.S3). A e
1h o exposu e, HA-Man NCs we e ound o be in e nalized
by mac ophages o bo h pheno ypes in a g ea e ex en han
non- unc ionalized HA NCs (Fig.6A). O no e, he inc eased
up ake was pa icula ly ema kable o M2 mac ophages
(3.1- old) (Fig.6A). These esul s we e alida ed by con o-
cal mic oscopy, con i ming he in e naliza ion o he NCs
and showing he same pa e n o he up ake o HA-Man
NCs e sus HA NCs by M1 and M2 mac ophages (Fig.6B).
These in e naliza ion assays using p ima y human mac-
ophages showed ha he unc ionaliza ion o HA wi h man-
nose g ea ly enhanced he abili y o he HA NCs o a ge
M2 mac ophages ( h ee old highe o M2 e sus M1). Simi-
la esul s ha e been epo ed o o he ypes o nanoca ie s
unc ionalized wi h mannose, al hough he imp o emen o
Table 3 DLS cha ac e iza ion o HA SD-NCs and HA-Man SD-NCs
Nanoca ie Pa icle size (nm) PDI Ze a po en ial (mV)
HA SD-NCs 160 ± 4 0.10 − 50 ± 7
HA-Man SD-NCs 158 ± 12 0.15 − 39 ± 7
Table 4 NTA cha ac e iza ion
o HA SD-NCs and HA-Man
SD-NCs
D10/50/90 dis ibu ion o he 10%/50%/90% o he pa icle popula ion, DLS dynamic ligh sca e ing, HA
SD-NCs / HA-Man SD-NCs nanocapsules p epa ed by sol en displacemen wi h a mannose-modi ied
50-kDa hyalu onic acid polyme ic coa ing and an oily co e o cap ylic-cap ic iglyce ides, NTA nanopa i-
cle acking analysis, PDI polydispe si y index, SD s anda d de ia ion
Nanoca ie Mean (nm) SD (nm) D10 (nm) D50 (nm) D90 (nm) Pa icles pe mL
HA SD-NCs 97 ± 8 27 70 94 132 4·1012 ± 2·1012
HA-Man SD-NCs 117 ± 7 39 80.7 110 164 4·1012 ± 2·1012
D ug Deli e y and T ansla ional Resea ch
1 3
58. Miz ahy S, Raz SR, Hasgaa d M, Liu H, So e -Tsu N, Cohen K,
D ash R, Landsman-Milo D, B eme MGEG, Moghimi SM, Pee
D. Hyalu onan-coa ed nanopa icles: he in luence o he molecu-
la weigh on CD44-hyalu onan in e ac ions and on he immune
esponse. J Con ol Release. 2011;156:231–8. h ps:// doi. o g/ 10.
1016/j. jcon el. 2011. 06. 031.
59. Ren T, Dexi L. Syn hesis o a ge able ca ionic amphiphiles. Te -
ahed on Le . 1999;40:7621–5. h ps:// doi. o g/ 10. 1016/ S0040-
4039(99) 01558-0.
60. Ryde g en S. Chemical modi ica ions o hyalu onan using
DMTMM-ac i a ed amida ion. 2013. h p:// www. ekna . uu. se/
s ude n . Accessed 30 May2022.
61. Gao S, Liu Y, Liu M, Yang D, Zhang M, Shi K. Biodeg adable
mesopo ous nanocomposi es wi h dual- a ge ing unc ion o
enhanced an i- umo he apy. J Con ol Release. 2022;341:383–
98. h ps:// doi. o g/ 10. 1016/J. JCONR EL. 2021. 11. 044.
62. Genna i A, Pelliccia M, Donno R, Kimbe I, Ti elli N. Mannosyla-
ion allows o syne gic (CD44/C- ype lec in) up ake o hyalu onic
acid nanopa icles in dend i ic cells, bu only upon co ec ligand
p esen a ion. Ad Heal hc Ma e . 2016;5:966–76. h ps:// doi. o g/
10. 1002/ ADHM. 20150 0941.
63. Maho S, Dash BC, O’Conno S, Pandi A. Mannosyla ed polye h-
yleneimine-hyalu onan nanohyb ids o a ge ed gene deli e y o
mac ophage-like cell lines. Bioconjug Chem. 2012;23:1138–48.
h ps:// doi. o g/ 10. 1021/ BC200 599K.
64. Lucas AT, Madden AJ, Zamboni WC. Challenges in p eclini-
cal o clinical ansla ion o an icance ca ie -media ed agen s.
Wiley In e discip Re Nanomed Nanobio echnol. 2016;8:642–53.
h ps:// doi. o g/ 10. 1002/ WNAN. 1394.
65. Wilhelm S, Ta a es AJ, Dai Q, Oh a S, Aude J, D o ak HF, Chan
WCW. Analysis o nanopa icle deli e y o umou s. Na Re
Ma e . 2016;1:16014. h ps:// doi. o g/ 10. 1038/ na e ma s. 2016. 14.
66. Tsoi KM, MacPa land SA, Ma X-Z, Spe zle VN, Eche e i J,
Ouyang B, Fadel SM, Sykes EA, Golda acena N, Ka hs JM, Con-
neely JB, Alman BA, Selzne M, Os owski MA, Adeyi OA, Zilman
A, McGil ay ID, Chan WCW. Mechanism o ha d-nanoma e ial
clea ance by he li e . Na Ma e . 2016;15:1212–21. h ps: // doi. o g/
10. 1038/ nma 4 718.
67. Saunde s NRM, Paolini MS, Fen on OS, Poul L, De allie e J,
Mpambani F, Da mon A, Be gè e M, Jibaul O, Ge main M,
Lange R. A nanop ime o imp o e he sys emic deli e y o
siRNA and mRNA. Nano Le . 2020;20:4264–9. h ps:// doi. o g/
10. 1021/ acs. nanol e . 0c007 52.
68. Saunde s NRM, Paolini MS, Fen on OS, Poul L, De allie e J,
Mpambani F, Da mon A, Be gè e M, Jibaul O, Ge main M, Lange
R, Lange R, Lange R, Lange R. A nanop ime o imp o e he sys-
emic deli e y o siRNA and mRNA. Nano Le . 2020;20:4264–9.
h ps:// doi. o g/ 10. 1021/ ACS. NANOL ETT. 0C007 52.
Publishe 's No e Sp inge Na u e emains neu al wi h ega d o
ju isdic ional claims in published maps and ins i u ional a ilia ions.