pha maceu ics
A icle
Dual-Ta ge ed Hyalu onic Acid/Albumin Micelle-Like
Nanopa icles o he Vec o iza ion o Doxo ubicin
Manuela Cu cio 1,*,† , Luis Diaz-Gomez 2,† , Giuseppe Ci illo 1, Fio e Pasquale Nicole a 1,
An onella Leggio 1and F ancesca Iemma 1
Ci a ion: Cu cio, M.; Diaz-Gomez, L.;
Ci illo, G.; Nicole a, F.P.; Leggio, A.;
Iemma, F. Dual-Ta ge ed Hyalu onic
Acid/Albumin Micelle-Like
Nanopa icles o he Vec o iza ion o
Doxo ubicin. Pha maceu ics 2021,13,
304. h ps://doi.o g/10.3390/
pha maceu ics13030304
Academic Edi o : Emanuela
Fabiola C apa o
Recei ed: 29 Janua y 2021
Accep ed: 23 Feb ua y 2021
Published: 26 Feb ua y 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Depa men o Pha macy, Heal h and Nu i ional Sciences, Uni e si y o Calab ia, 87036 Rende (CS), I aly;
[email p o ec ed] (G.C.); [email p o ec ed] (F.P.N.); [email p o ec ed] (A.L.);
[email p o ec ed] (F.I.)
2Depa amen o de Fa macología, Fa macia y Tecnología Fa macéu ica, I+D Fa ma G oup, Facul ad de
Fa macia and Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS), Uni e sidade de San iago de
Compos ela, 15782 San iago de Compos ela, Spain; [email p o ec ed]
*Co espondence: manuela.cu [email p o ec ed]; Tel.: +39-0984493011
† These au ho s con ibu e equally o his pape .
Abs ac :
D ug a ge ing o umo cells is one o he g ea challenges in cance he apy; nanopa icles
based on na u al polyme s ep esen aluable ools o achie e his aim. The abili y o espond
o en i onmen al signals om he pa hological si e (e.g., al e ed edox po en ial), oge he wi h
he speci ic in e ac ion wi h memb ane ecep o s o e exp essed on cance cells memb ane (e.g.,
CD44 ecep o s), ep esen he main ea u es o ac i ely a ge ed nanopa icles. In his wo k, edox-
esponsi e micelle-like nanopa icles we e p epa ed by sel -assembling o a hyalu onic acid–human
se um albumin conjuga e con aining cys amine moie ies ac ing as a unc ional space . The conjuga ion
p ocedu e consis ed o a educ i e amina ion s ep o hyalu onic acid ollowed by condensa ion wi h
albumin. A e sel -assembling, nanopa icles wi h a mean size o 70 nm and able o be des abilized
in educing media we e ob ained. Doxo ubicin-loaded nanopa icles modula ed d ug elease a e
in esponse o di e en edox condi ions. Finally, he iabili y and up ake expe imen s on heal hy
(BALB-3T3) and me as a ic cance (MDA-MB-231) cells p o ed he po en ial applicabili y o he
p oposed sys em as a d ug ec o in cance he apy.
Keywo ds:
hyalu onic acid; human se um albumin; polysaccha ide-p o ein conjuga e; micelle-like
nanopa icles; edox- esponsi e; ac i e a ge ing; cance he apy
1. In oduc ion
Non-speci ic a ge ing and mul i-d ug esis ance a e he main limi a ions o he suc-
cess o con en ional chemo he apy. In he las decades, o o e come hese p oblems,
nanopa icle o mula ions ha e been p oposed as aluable ools o deli e con en ional
d ugs o he si e o in e es , modi ying hei pha macokine ics and he apeu ic esponse [
1
].
The sui abili y o nanopa icles in cance ea men is ela ed o some e idence. Nanopa -
icles can e icien ly accumula e in he umo si e by i ue o angiogenesis and he lack
o lympha ic d ainage, a condi ion known as he enhanced pe mea ion and e en ion
(EPR) e ec [
2
]. In addi ion, he possibili y o modi y he physicochemical and su ace
p ope ies o nanopa icles allows he amoun o d ug deli e ed o he a ge si e o be
ema kably inc eased. Fo his pu pose, wo di e en app oaches can be ollowed: (i)
conjuga ion wi h ligands binding o ecep o s o e -exp essed on o he umo cells [
3
]; (ii)
unc ionaliza ion wi h chemical g oups able o espond o signals om umo mic oen-
i onmen s [
4
,
5
]. These enginee ed ma e ials, known as ac i ely- a ge ed d ug deli e y
sys ems, ha e eme ged as “magic bulle s” able o hi he si e o disease, a oiding he side
e ec s o o he heal hy o gans [
6
]. Small molecules [
7
] o mac omolecula compounds [
8
]
a e employed as ac i e a ge ing ligands, whe eas he a ia ion o pH, edox po en ial,
Pha maceu ics 2021,13, 304. h ps://doi.o g/10.3390/pha maceu ics13030304 h ps://www.mdpi.com/jou nal/pha maceu ics
Pha maceu ics 2021,13, 304 2 o 16
empe a u e o eac i e oxygen species concen a ion a e he mos exploi ed en i onmen al
s imuli in a umo - a ge ed elease [
9
]. Fo example, i is well known ha he ema kable
di e ences in glu a hione (GSH) concen a ion in cance cells (2–10 mM), compa ed o
he no mal ex acellula ma ix (2–20
µ
M), gene a e a high edox po en ial [
10
]. Thus,
by inse ing in he nanopa icle s uc u e edox- esponsi e unc ionali ies, i.e., disul ide
linkages [
11
], ma e ials able o selec i ely des abilize inside cells and elease he payload
can be achie ed.
To da e, he wide a ailabili y o ma e ials and p epa a ion me hods allows o a la ge
numbe o nanopa icle a chi ec u es o be ob ained endowed wi h di e en a ge ing lig-
ands and s imuli- esponsi e chemical g oups. Na u al polyme s, such as polysaccha ides
and p o eins, ha e eme ged as p omising base ma e ials o d ug deli e y pla o ms in
cance he apy due o hei unique physicochemical and biological p ope ies such as non-
immunogenici y, biocompa ibili y, biodeg adabili y, and he p esence o se e al unc ional
g oups a ailable o chemical modi ica ion [
12
]. P o ein nanopa icles can be p epa ed
unde mild condi ions wi hou he use o oxic chemicals o o ganic sol en s, allowing
o easily con ol he pa icle size [
13
,
14
]; on he o he hand, polysaccha ide ma e ials a e
sa e and may con e “s eal h” p ope ies o he inal nanopa icle sys em, dec easing he
up ake by he mononuclea phagocy e sys em and enhancing he blood ci cula ion ime
and he possibili y o accumula ion in he disease si e [
15
]. These aluable p ope ies ha e
inspi ed he p epa a ion o p o ein-polysaccha ide conjuga es showing supe io ea u es
no a ainable using he indi idual componen [16,17].
Human se um albumin (HSA) is he mos abundan p o ein in he blood and one o
he mos a ac i e ma e ials o he de elopmen o no el nanomedicines [
18
,
19
] due o i s
high d ug-loading abili y, sel -assembling p ope ies, and long hal -li e. These p ope ies,
among o he s, like i s lack o oxici y and immunogenici y and i s p e e en ial up ake in
umo and in lamed issues, make HSA a p omising candida e o d ug deli e y. Fu -
he mo e, he e ia y s uc u e o HSA con ains a d ug-binding si e
(subdomain I B)
ha
has a ema kable a ini y o doxo ubicin, among o he an i umo d ugs [
20
,
21
]. The in-
c eased hyd ophobici y o HSA a e binding be ween doxo ubicin signi ican ly enhances
he sel -assembly p ope ies o he p o ein [
22
]. HSA nanopa icles ha e shown ac i e
umo a ge ing ia ecep o -media ed endocy osis, including GP60 and neona al Fc e-
cep o s ha a e o e exp essed in umo cells. Mo eo e , ecen s udies ha e epo ed
ha HSA nanopa icles unde go agg ega ion in a umo pH en i onmen , hus a o ing
accumula ion and e en ion o HSA-d ug complexes in umo issues [23].
Ano he app oach o achie e ac i e d ug a ge ing is he inco po a ion o hyalu onic
acid (HA) in o nanopa icle sys ems. HA is a nega i ely cha ged, non-sul a ed glycosamino-
glycan composed o epea ing uni s o D-glucu onic acid and N-ace yl-D-glucosamine
bound by be a-linkages and is ound h oughou he body in he ex acellula ma ix and
syno ial luids [
24
]. HA-based nanopa icles ha e been ex ensi ely explo ed as deli e y
de ices in cance he apy by i ue o hei abili y o speci ically bind CD44 ecep o o e ex-
p essed on a ious cance cells [
25
]. Fu he mo e, i is possible o endo se HA nanopa icles
wi h edox- esponsi e p ope ies by cys amine deco a ion, enhancing he desi ed a ge ing
p ope ies o issues exhibi ing inc eased GSH le els, such as umo s [26].
In his wo k, we aimed o de elop a no el d ug deli e y sys em wi h edox- esponsi e
and ac i ely a ge ing p ope ies o an icance he apies. Micelle-like nanopa icles we e
ob ained by a simple sel -assembling p ocess o a conjuga e (HAssHSA) composed o
cys amine-modi ied HA (HAcys) and HSA. We hypo hesized ha he bioconjuga ion o
HAssHSA and he esul ing sel -assembling nanopa icles (HNPs) could me ge he ad-
an ageous ea u es o each componen in a nanopa icle sys em wi h enhanced a ge ing
p ope ies o CD44-o e exp essing cells, edox esponsi eness, and high loading abili y.
The ob ained nanopa icles we e cha ac e ized by dynamic ligh sca e ing (DLS) and
ansmission elec on mic oscopy (TEM), and edox- igge ed des abiliza ion assays we e
pe o med by measu ing he a ia ion o he nanopa icle mean diame e in educing
media. The sui abili y o HNPs as d ug ca ie s was hen e alua ed by loading wi h dox-
Pha maceu ics 2021,13, 304 3 o 16
o ubicin hyd ochlo ide (DOX), a DNA opoisome ase II inhibi o wi h a b oad-spec um
an ineoplas ic ac i i y [
27
]. Howe e , DOX use in clinical p ac ice is o en limi ed by
se e al side e ec s.
In i o
elease p o iles om DOX-loaded HNPs we e in es iga ed,
a ying he edox po en ial o he su ounding medium. Finally, cy o oxici y and cellula
up ake expe imen s we e pe o med on heal hy and cance cells o assess he sa e y he
po en ial sui abili y o he sys em in cance he apy.
2. Ma e ials and Me hods
2.1. Syn hesis o HAcys
HAcys was ob ained by educ i e amina ion [
28
,
29
]. B ie ly, a e 0.1 g (0.24 mmol
disaccha ide epea ing uni s) o HA (10 kDa) we e dissol ed in 4 mL o an H
2
O:DMSO
(3:7 / ) mix u e, 0.31 g (4.93 mmol) o sodium cyanobo ohyd ide and 1.1 g (4.45 mmol)
o cys amine dihyd ochlo ide (cysHCl) we e added, and he mix u e was s i ed o 24 h
a oom empe a u e. The esul ing solu ion was pu i ied using dialysis memb anes wi h
molecula weigh cu o (MWCO) o 3.5 kDa agains wa e a 20
◦
C o 72 h, and inally
eeze-d ied (98% yield).
1
H-NMR spec a we e eco ded on a B uke A ance 300 (B uke
I aly, Milan, I aly) a 25 ◦C using D2O as a sol en .
HA was pu chased om Li eco e Biomedical (Chaska, MN, USA). All o he chemicals
we e pu chased om Me ck/Sigma-Ald ich, Da ms ad , Ge many. Dialysis memb anes
we e pu chased om Medicell In e na ional LTD (London, UK).
2.2. Syn hesis o HAssHSA Conjuga e
HSA (0.074 g) and 1-e hyl-3-(3-dime hylaminop opyl) ca bodiimide (EDC) (0.010 g,
0.052 mmol) we e dissol ed in 4 mL o a phospha e bu e solu ion (0.01 M, pH 7.4) and
le o eac o 1 h a oom empe a u e unde magne ic s i ing. Then, HAcys
(0.017 g)
dissol ed in 2 mL o phospha e bu e (0.01 M, pH 7.4) was added. The mix u e was
magne ically s i ed o 24 h a oom empe a u e, pu i ied by dialysis (MWCO 12–14 kDa)
agains wa e a 20
◦
C o 72 h, and inally eeze-d ied (98% yield).
1
H-NMR spec a
we e eco ded on a B uke A ance 300 (B uke I aly, Milan, I aly) a 25
◦
C using D
2
O
as a sol en .
All chemicals we e pu chased om Me ck/Sigma-Ald ich, Da ms ad , Ge many.
Dialysis memb anes we e pu chased om Medicell In e na ional LTD (London, UK).
2.3. P epa a ion o Labeled HAssHSA Conjuga e
Fo he syn hesis o he labeled conjuga e, luo escein iso hiocyana e (FITC) was
co alen ly linked o he HAssHSA as p e iously desc ibed [
30
]. HAssHSA (60 mg) was
dissol ed in 3.0 mL sodium ca bona e bu e (100 mM, pH 9.2), and hen 0.6 mL FITC
solu ion in sodium ca bona e bu e (1.0 mg mL−1) was added and allowed o eac a RT
o 3 h. The ob ained FITC-HAssHSA was pu i ied by dialysis (MWCO 12–14 kDa) agains
5.0 mM phospha e bu e (pH 3.0) o wo days and cha ac e ized by UV–Vis spec a.
UV–Vis analysis was pe o med on an E olu ion 201 spec opho ome e (The mo Fishe
Scien i ic, Hillsbo o, OR, USA) ope a ing wi h 1.0 cm qua z cells. All chemicals we e
pu chased om Me ck/Sigma-Ald ich, Da ms ad , Ge many.
2.4. De e mina ion o he C i ical Agg ega ion Concen a ion (CAC)
The modi ica ion o he luo escence p ope ies o he nonpola p obe py ene, when
loca ed inside a micelle hyd ophobic co e, was exploi ed o measu e he CAC o HAssHSA
in he aqueous phase [
31
,
32
]. In de ail, 20.0
µ
L py ene solu ion a a concen a ion o
3.0 ×10−5M
in ace one was e apo a ed in ials. HAssHSA conjuga e was dissol ed
a concen a ions anged om 1.6
×
10
−7
o 1 mg mL
−1
in phospha e bu e (0.01 M,
pH 7.4
) unde magne ic s i ing, and hen 1 mL o each solu ion was added o he py ene
ials. The con en s o he ials we e mixed o 12 h, he eby leading o solu ions wi h a
py ene concen a ion o ca. 6.0
×
10
−7
M. Then, he in ensi y a ios (I
3
/I
1
) o he hi d
ib onic band a 385 nm o he i s one a 373 nm o he luo escence emission spec a
Pha maceu ics 2021,13, 304 4 o 16
o py ene we e eco ded a 25
◦
C. Py ene luo escence emission spec a (
λexc
= 336 nm;
λem = 350–500 nm
) we e eco ded on Hi achi F-2500 spec ome e (Tokyo, Japan). All
chemicals we e pu chased om Me ck/Sigma-Ald ich, Da ms ad , Ge many.
2.5. P epa a ion and Cha ac e iza ion o Micelle-Like Nanopa icles
Emp y nanopa icle HNPs we e ob ained by dispe sing he HAssHSA conjuga e
( inal concen a ion o 1 mg mL
−1
) in phospha e bu e solu ion (0.01 M, pH 7.4) unde
magne ic s i ing o 2 h a oom empe a u e. Fo he cell up ake expe imen s, FITC-
HAssHSA was ea ed in he same manne , ob aining he luo escen -labeled FITC-HNPs.
The size dis ibu ion o HNPs ( inal concen a ion 1 mg mL
−1
) was de e mined using
a 90 Plus pa icle size analyze DLS equipmen (B ookha en Ins umen s Co po a ion,
Hol s ille, NY, USA) a 25
◦
C. The au oco ela ion unc ion was measu ed a 90
◦
, and he
lase beam ope a ed a 658 nm. The polydispe si y index (PDI) was di ec ly ob ained om
he ins umen al da a i ing p ocedu es by he in e se Laplace ans o ma ion and Con in
me hods. PDI alues
≤
0.3 indica e homogenous and mono-dispe se popula ions [
33
].
Mo phological analysis o esicles was ca ied ou using TEM (HRTEM/Tecnai F30
(80 kV)
FEI Company, Hillsbo o, OR, USA). A d op o he esicle dispe sion was placed on a Cu
TEM g id (200 mesh, Plano GmbH, We zla , Ge many), and he sample in excess was
emo ed using a piece o il e pape . A d op o 2% (w/ ) phospho ungs ic acid solu ion
was hen deposi ed on he ca bon g id o 2 min. Once he excess s aining agen was
emo ed wi h il e pape , he samples we e ai -d ied, and he hin ilm o s ained esicles
was obse ed.
All chemicals we e pu chased om Me ck/Sigma-Ald ich, Da ms ad , Ge many.
2.6. Des abiliza ion Expe imen s
Des abiliza ion expe imen s o emp y nanopa icles in educ i e en i onmen s we e
pe o med by he dialysis me hod. B ie ly, in sepa a e expe imen s, 4 mL o eshly
p epa ed emp y nanopa icles ( inal concen a ion 1 mg mL
−1
) we e loaded in a dialysis
bag (MWCO 12–14 kDa) and dialyzed agains 30 mL phospha e bu e (0.01 M, pH 7.4)
con aining GSH a di e en concen a ions (0 mM and 10 mM) a 37
◦
C in a beake wi h
cons an s i ing. A e 24 h, he mean diame e and PDI we e measu ed by DLS. Each
analysis was pe o med in iplica e.
2.7. DOX Loading
DOX-loaded nanopa icles (DOX@HNPs) we e p epa ed by dispe sing HAssHSA
conjuga e ( inal concen a ion 1 mg mL
−1
) in a 58.8
µ
M DOX hyd ochlo ide solu ion in
phospha e bu e (0.01 M, pH 7.4) unde magne ic s i ing o 12 h a oom empe a u e [
34
].
The dispe sion was used as such in he nex elease expe imen s. The loading e iciency
was con i med by dilu ing 1 mL o DOX@HNPs dispe sion in 25 mL o me hanol in o de
o dis up he micelle-like s uc u e [
35
], ollowed by he measu emen o he luo escence
o he solu ion (λexc = 480 nm; λem = 590 nm).
2.8. Release Expe imen s
Release expe imen s we e ca ied ou by means o a dialysis me hod unde sink condi-
ions. In wo di e en expe imen s, 2 mL DOX@HNPs dispe sion (polyme concen a ion
1 mg mL
−1
) we e loaded in a dialysis bag (MWCO 3.5 kDa) and dialyzed agains 10 mL
phospha e (0.01 M, pH 7.4) bu e con aining GSH a di e en concen a ions (0 mM and
10 mM
) a 37
◦
C in a beake wi h cons an s i ing. A p e-es ablished imes, samples
(0.5 mL)
o elease medium we e wi hd awn, eplaced wi h esh medium and quan i ied
by a luo escence spec ome e (
λexc
= 480 nm;
λem
= 590 nm) using a s anda d calib a ion
cu e o and DOX (2–30
µ
M) p epa ed unde he same condi ions. Expe imen s we e
pe o med in iplica e.
Pha maceu ics 2021,13, 304 5 o 16
2.9. Cy o oxici y Expe imen s
MDA-MB231 (Cell Biolabs; San Diego, CA, USA) and BALB/3T3 (CCL-163; ATCC,
Manassas, VA, USA) cell lines we e main ained in DMEM supplemen ed wi h 10 / %
FBS and 1% an ibio ics (10,000 U/mL penicillin and 10,000
µ
g/mL s ep omycin) in an
incuba o a 37 ◦C and 5% CO2.
The cy o oxic e ec s o he d ug, emp y nanopa icles and d ug-loaded sys ems on
cell iabili y we e de e mined using a Cell Coun ing Ki 8 (CCK-8; Dojindo Molecula
Technologies; Rock ille, MD, USA). MDA-MB231 o BALB/3T3 cells we e seeded in 96-
well pla es (1
×
10
4
cells/well) and incuba ed o e nigh . Then, cells we e exposed o
inc easing concen a ion o he d ug (0.001 o 5
µ
g mL
−1
) o HNPs (0.1 o 2 mg mL
−1
)
and u he incuba ed o 24 o 48 h. A each ime poin , he medium was disca ded, cells
we e washed wi h PBS, and 100
µ
L o eshly p epa ed CCK-8 wo king solu ion (90
µ
L
o cul u e medium and 10
µ
L o CCK-8 eagen ) we e added o each well and incuba ed
o 2 h a
37 ◦C
. Finally, abso bance (450 nm) was eco ded using a mic opla e eade
(Model 8; Bio-Rad, Philadelphia, PA, USA). Cell iabili y was de e mined by compa ing
he abso bance o each condi ion wi h a posi i e con ol consis ing o cells incuba ed in he
cul u e medium.
DOX@HNPs cy o oxici y was e alua ed in MDA-MB231 and BALB/3T3 cells. Fo he
p epa a ion o DOX-loaded HNPs, 1 mg o HAssHSA was dispe sed in 5 mL o eshly
p epa ed DOX solu ions (40, 20, 10, and 5
µ
g mL
−1
) in phospha e bu e unde magne ic
s i ing o e nigh . Then, 1 mL o each nanopa icle dispe sion was added o 3 mL o
phospha e bu e in o de o ob ain a inal nanopa icle concen a ion o 0.025 mg mL
−1
and DOX concen a ions o 5, 2.5, 1.25, and 0.625
µ
g mL
−1
. MDA-MB231 o BALB/3T3
cells (1
×
10
4
cells pe well) we e seeded in o 96-well pla es and incuba ed o 24 h as
explained be o e. Then, he medium was emo ed, and 100
µ
L o esh medium and
100 µL
o he eshly p epa ed DOX@HNPs nanopa icle solu ions we e added o each well and
cul u ed o 24 o 48 h. Phospha e bu e (0.01 M, pH 7.4) was used as he nega i e con ol.
A each ime poin , he medium was disca ded, cells we e washed wi h PBS and 100
µ
L
o eshly p epa ed solu ion (90
µ
L o cul u e medium and 10
µ
L o CCK-8 eagen we e
added o each well and incuba ed o 3 h a 37
◦
C. Abso bance (450 nm) was hen eco ded
using a Model 8 mic opla e eade (Bio-Rad, USA).
2.10. Visualiza ion o CD44-Media ed Cellula Up ake
MDA-MB231 and BALB/3T3 cells we e cul u ed as explained in Sec ion 2.6 and
seeded in 8-well glass slides (Lab-Tek II chambe slide; The mo Scien i ic, Wal ham, MA,
USA) a a concen a ion o 5
×
10
4
cells/well. A e 24 h o incuba ion, cells we e washed
wi h PBS and hen, CD44 ecep o s we e blocked by incuba ing cells wi h 10 equi alen s o
ee HA (solu ion in cul u e medium) o 1 h. Un ea ed cells we e used as posi i e con ols.
Subsequen ly, cells we e washed wi h PBS and ea ed wi h FITC-HNPs (0.1 mg mL
−1
)
o 24 h. A each ime poin , cells we e washed wi h PBS, ixed wi h pa a o maldehyde
4% (
100 µL
pe well) o 10 min, washed h ee imes again wi h PBS and hen, incuba ed
wi h T i on X-100 (0.2% in PBS pH 7.4, 40
µ
L, 5 min), and washed h ee imes again wi h
PBS. Then, cells we e s ained wi h a phalloidin dye (Alexa Fluo 488 phalloidin, Molecula
P obes; Eugene, OR, USA) o 20 min ollowing he manu ac u e ’s p o ocol and washed
again wi h PBS. Subsequen ly, samples we e moun ed using DAPI P oLong gold (Molecula
P obes; Eugene, OR, USA), co e ed wi h a co e glass, and ozen un il obse a ion.
Con ocal images we e eco ded using a Leica con ocal TCS-SP5 (Leica Mic osys ems;
We zla , Ge many). The e alua ion o cellula up ake o DOX-loaded HNPs (DOX@HNPs)
was also ca ied ou using con ocal mic oscopy. Cells we e seeded, and CD44 ecep o s
blocked, as explained be o e. Following his, cells we e ea ed wi h cul u e medium
(nega i e con ol), a 0.8 mg mL
−1
DOX solu ion o DOX@HNPs (con aining 0.8 mg mL
−1
o DOX), and incuba ed o 12 h. Finally, cells we e ixed and s ained as desc ibed be o e
and obse ed using a Leica con ocal TCS-SP5 (Leica Mic osys ems; We zla , Ge many).
Pha maceu ics 2021,13, 304 6 o 16
2.11. S a is ical Analysis
S a is ical analyses we e analyzed using G aphPad P ism (G aphPad So wa e, La
Jolla, CA, USA). All esul s we e exp essed as mean alues wi h s anda d de ia ions. Two-
way analysis o a iance and Tukey’s mul iple compa ison pos - es we e used. Di e ences
we e conside ed signi ican o p< 0.05.
3. Resul s and Discussion
3.1. Syn hesis o HAssHSA Conjuga e
The co alen conjuga ion o biomac omolecules is a ac ing inc easing in e es be-
cause o he possibili y o combine he ad an ages o each componen , minimizing hei
espec i e liabili ies [
36
]. HAssHSA conjuga e was ob ained in a wo-s ep p ocedu e con-
sis ing in (i) he syn hesis o HAcys by educ i e amina ion, a selec i e, as and easy
eac ion p ocedu e using cysHCl and sodium cyanobo ohyd ide as eac an s; and (ii) he
conjuga ion o HAcys wi h HSA ia co alen coupling wi h EDC (Figu e 1).
Pha maceu ics 2021, 13, x 6 o 17
CD44 ecep o s blocked, as explained be o e. Following his, cells we e ea ed wi h cul-
u e medium (nega i e con ol), a 0.8 mg mL
−1
DOX solu ion o DOX@HNPs (con aining
0.8 mg mL
−1
o DOX), and incuba ed o 12 h. Finally, cells we e ixed and s ained as de-
sc ibed be o e and obse ed using a Leica con ocal TCS-SP5 (Leica Mic osys ems; We z-
la , Ge many).
2.11. S a is ical Analysis
S a is ical analyses we e analyzed using G aphPad P ism (G aphPad So wa e, La
Jolla, CA, USA). All esul s we e exp essed as mean alues wi h s anda d de ia ions.
Two-way analysis o a iance and Tukey’s mul iple compa ison pos - es we e used. Di -
e ences we e conside ed signi ican o p < 0.05.
3. Resul s and Discussion
3.1. Syn hesis o HAssHSA Conjuga e
The co alen conjuga ion o biomac omolecules is a ac ing inc easing in e es be-
cause o he possibili y o combine he ad an ages o each componen , minimizing hei
espec i e liabili ies [36]. HAssHSA conjuga e was ob ained in a wo-s ep p ocedu e con-
sis ing in (i) he syn hesis o HAcys by educ i e amina ion, a selec i e, as and easy e-
ac ion p ocedu e using cysHCl and sodium cyanobo ohyd ide as eac an s; and (ii) he
conjuga ion o HAcys wi h HSA ia co alen coupling wi h EDC (Figu e 1).
Figu e 1. Syn hesis o HAcys and HAssHSA conjuga e.
HAcys and HAssHSA we e cha ac e ized by FT-IR and
1
H-NMR analyses. As de-
pic ed in Figu e 2a, he FT-IR spec um o HAcys did no show any signi ica i e a ia ion
compa ed o na i e HA because he abso p ion bands o cys amine we e o e shadowed
by he mo e in ense abso p ion bands o he polysaccha ide. In he
1
H-NMR spec a (Fig-
u e 2b), he signals o he wo HA anome ic p o ons (β and β’) we e e iden a a ound
4.30 ppm, while he p esence o cys amine esidues was quan i a i ely de e mined om
ela i e in eg a ion o peak (α) a 1.81 ppm co esponding o he h ee me hyl p o ons o
N-ace yl g oup o HA and he iple signal (γ) a 2.80 ppm ela ed o he ou me hylene
p o ons o cys amine. The de i a iza ion deg ee (DD), exp essed as cys amine moie ies
wi h espec o HA epea ing uni s, was calcula ed o be 12.7%.
Figu e 1. Syn hesis o HAcys and HAssHSA conjuga e.
HAcys and HAssHSA we e cha ac e ized by FT-IR and
1
H-NMR analyses. As de-
pic ed in Figu e 2a, he FT-IR spec um o HAcys did no show any signi ica i e a ia ion
compa ed o na i e HA because he abso p ion bands o cys amine we e o e shadowed
by he mo e in ense abso p ion bands o he polysaccha ide. In he
1
H-NMR spec a
(Figu e 2b)
, he signals o he wo HA anome ic p o ons (
β
and
β
’) we e e iden a a ound
4.30 ppm, while he p esence o cys amine esidues was quan i a i ely de e mined om
ela i e in eg a ion o peak (
α
) a 1.81 ppm co esponding o he h ee me hyl p o ons o
N-ace yl g oup o HA and he iple signal (
γ
) a 2.80 ppm ela ed o he ou me hylene
p o ons o cys amine. The de i a iza ion deg ee (DD), exp essed as cys amine moie ies
wi h espec o HA epea ing uni s, was calcula ed o be 12.7%.
The FT-IR spec um o HAssHSA conjuga e showed he ypical abso p ion bands o
HA (1640 and 1032 cm
−1
), co esponding o he s e ching ib a ions o amide ca bonyl
and C–OH, espec i ely). An abso p ion band a 1557 cm
−1
asc ibable o he seconda y
amide band o HSA was also p esen in he HAssHSA spec um, con i ming he success ul
polysaccha ide-p o ein conjuga ion. Fu he mo e, he conjuga ion was also e idenced
by he p esence o obse able peaks in he a oma ic egion (8.50–6.50 ppm) and alipha ic
egion (0.50–1.80 ppm) o he 1H-NMR spec um o HAssHSA.
The sel -assembling capabili y o he HAssHSA conjuga e was e alua ed by measu ing
i s CAC, using py ene as a luo escen p obe o hyd ophobic domain o ma ion. The
de e mina ion o CAC is pi o al in iew o a po en ial applica ion
in i o
, whe e he sys em
is highly dilu ed in he sys emic ci cula ion: he lowe he CAC alue, he g ea e he s abili y
o he conjuga e ha will be able o o ganize a low concen a ions. Fo his measu emen ,
he dependence o py ene luo escence spec a (I
384
/I
373
a io) on he loga i hm o conjuga e
concen a ion was conside ed, and he esul s a e depic ed in Figu e 3.
Pha maceu ics 2021,13, 304 7 o 16
Pha maceu ics 2021, 13, x 7 o 17
(a) (b)
Figu e 2. FT-IR (a) and
1
H-NMR (b) spec a o HSA, HA, HAcys and HAssHSA.
The FT-IR spec um o HAssHSA conjuga e showed he ypical abso p ion bands o
HA (1640 and 1032 cm
−1
), co esponding o he s e ching ib a ions o amide ca bonyl
and C–OH, espec i ely). An abso p ion band a 1557 cm
−1
asc ibable o he seconda y
amide band o HSA was also p esen in he HAssHSA spec um, con i ming he success-
ul polysaccha ide-p o ein conjuga ion. Fu he mo e, he conjuga ion was also e idenced
by he p esence o obse able peaks in he a oma ic egion (8.50–6.50 ppm) and alipha ic
egion (0.50–1.80 ppm) o he
1
H-NMR spec um o HAssHSA.
The sel -assembling capabili y o he HAssHSA conjuga e was e alua ed by measu -
ing i s CAC, using py ene as a luo escen p obe o hyd ophobic domain o ma ion. The
de e mina ion o CAC is pi o al in iew o a po en ial applica ion in i o, whe e he sys-
em is highly dilu ed in he sys emic ci cula ion: he lowe he CAC alue, he g ea e he
s abili y o he conjuga e ha will be able o o ganize a low concen a ions. Fo his meas-
u emen , he dependence o py ene luo escence spec a (I
384
/I
373
a io) on he loga i hm o
conjuga e concen a ion was conside ed, and he esul s a e depic ed in Figu e 3.
Figu e 3. Dependence o py ene luo escence spec um signals on HAssHSA concen a ion a pH
7.4.
Figu e 2. FT-IR (a) and 1H-NMR (b) spec a o HSA, HA, HAcys and HAssHSA.
Pha maceu ics 2021, 13, x 7 o 17
(a) (b)
Figu e 2. FT-IR (a) and
1
H-NMR (b) spec a o HSA, HA, HAcys and HAssHSA.
The FT-IR spec um o HAssHSA conjuga e showed he ypical abso p ion bands o
HA (1640 and 1032 cm
−1
), co esponding o he s e ching ib a ions o amide ca bonyl
and C–OH, espec i ely). An abso p ion band a 1557 cm
−1
asc ibable o he seconda y
amide band o HSA was also p esen in he HAssHSA spec um, con i ming he success-
ul polysaccha ide-p o ein conjuga ion. Fu he mo e, he conjuga ion was also e idenced
by he p esence o obse able peaks in he a oma ic egion (8.50–6.50 ppm) and alipha ic
egion (0.50–1.80 ppm) o he
1
H-NMR spec um o HAssHSA.
The sel -assembling capabili y o he HAssHSA conjuga e was e alua ed by measu -
ing i s CAC, using py ene as a luo escen p obe o hyd ophobic domain o ma ion. The
de e mina ion o CAC is pi o al in iew o a po en ial applica ion in i o, whe e he sys-
em is highly dilu ed in he sys emic ci cula ion: he lowe he CAC alue, he g ea e he
s abili y o he conjuga e ha will be able o o ganize a low concen a ions. Fo his meas-
u emen , he dependence o py ene luo escence spec a (I
384
/I
373
a io) on he loga i hm o
conjuga e concen a ion was conside ed, and he esul s a e depic ed in Figu e 3.
Figu e 3. Dependence o py ene luo escence spec um signals on HAssHSA concen a ion a pH
7.4.
Figu e 3.
Dependence o py ene luo escence spec um signals on HAssHSA concen a ion a pH 7.4.
A low concen a ions, he in ensi y alues emained almos unchanged; when he
amoun o conjuga e inc eased, a sha p change o he in ensi y was obse ed, indica -
ing he onse o sel -assembly. F om he c osso e poin s, a CAC o 1.6
µ
g mL
−1
was
calcula ed in acco dance wi h he da a epo ed in he li e a u e o HA-based nanopa i-
cles
[37,38]
. I is well-known ha he amphiphilic p ope ies o HSA, a globula p o ein
wi h hyd ophobic domains in he inne co e and hyd ophilic domains in he su ace laye ,
enable sel -assembling in o micella s uc u es [
39
–
41
]. In ac , i was ecen ly epo ed ha
HA-albumin conjuga es could sel -assemble in o uni o m micelle-like s uc u es [42].
3.2. P epa a ion o HNPs and DOX Loading
HNPs we e p epa ed in a s aigh - o wa d p ocedu e by dispe sing he HAssHSA
conjuga e in phospha e bu e solu ion a pH 7.4, exploi ing he amphiphilic cha ac e o he
sys em. I was hypo hesized ha he hyd ophobic co e is cons i u ed by he hyd ophobic
domains o HSA, while he hyd ophilic HA chains o m he shell o he micelle-like
nanopa icles [42] (Figu e 4a).
Pha maceu ics 2021,13, 304 8 o 16
Pha maceu ics 2021, 13, x 8 o 17
A low concen a ions, he in ensi y alues emained almos unchanged; when he
amoun o conjuga e inc eased, a sha p change o he in ensi y was obse ed, indica ing
he onse o sel -assembly. F om he c osso e poin s, a CAC o 1.6 μg mL
−1
was calcula ed
in acco dance wi h he da a epo ed in he li e a u e o HA-based nanopa icles [37,38].
I is well-known ha he amphiphilic p ope ies o HSA, a globula p o ein wi h hyd o-
phobic domains in he inne co e and hyd ophilic domains in he su ace laye , enable
sel -assembling in o micella s uc u es [39–41]. In ac , i was ecen ly epo ed ha HA-
albumin conjuga es could sel -assemble in o uni o m micelle-like s uc u es [42].
3.2. P epa a ion o HNPs and DOX Loading
HNPs we e p epa ed in a s aigh - o wa d p ocedu e by dispe sing he HAssHSA
conjuga e in phospha e bu e solu ion a pH 7.4, exploi ing he amphiphilic cha ac e o
he sys em. I was hypo hesized ha he hyd ophobic co e is cons i u ed by he hyd o-
phobic domains o HSA, while he hyd ophilic HA chains o m he shell o he micelle-
like nanopa icles [42] (Figu e 4a).
(a) (b)
Figu e 4. (a) P oposed a chi ec u e o he esicle memb ane and (b) TEM image o nega i ely
s ained HNPs (20 K magni ica ion).
DLS analysis showed a unimodal pa icle size dis ibu ion, wi h a mean hyd ody-
namic diame e o 70 ± 9 nm and a PDI o 0.2, while TEM mic og aphs e ealed ha he
HNPs we e cha ac e ized by sphe ical shape and a mean diame e alue close o ha ec-
o ded by DLS (Figu e 4b). The size o he HNPs desc ibed in his wo k is signi ican ly
smalle compa ed o p e iously epo ed HSA nanopa icles p epa ed by he desol a ion
me hod [43–45] o sel -assembling p ocesses [46–48]. Pa icle size is he esul o he com-
bina ion o di e en pa ame e s, including physic-chemical p ope ies o he amphiphile,
p epa a ion me hod, and expe imen al condi ions, ha s ongly a ec he ea angemen
o he polyme s uc u e du ing he sel -assembling s ep. HNPs dimensions p esen an
ideal size ha is la ge enough o accumula e in umo issues ia enhanced pe meabili y
and e en ion (EPR) e ec . Howe e , since he pa icle size is lowe han 100 nm, i is ex-
pec ed o achie e ex ensi e umo pene a ion [49].
3.3. Des abiliza ion Expe imen s
GSH- esponsi e beha io o HNPs was assessed by means o des abiliza ion expe i-
men s pe o med in media mimicking he ex acellula and in acellula edox en i on-
men s. In pa icula , he a ia ion o he dimensional dis ibu ion o micelle-like nanopa -
icles was analyzed by DLS a e 24 h incuba ion in phospha e bu e a physiological pH
wi h o wi hou GSH 10 mM. The mean diame e o HNPs emained almos unchanged
in he medium, mimicking he ex acellula en i onmen , while in he p esence o he
ipep ide GSH bo h he mean diame e and he PDI d ama ically inc eased o 130 nm and
0.54, espec i ely. This obse a ion con i ms ha HNPs we e esponsi e o GSH, which
Figu e 4.
(
a
) P oposed a chi ec u e o he esicle memb ane and (
b
) TEM image o nega i ely s ained
HNPs (20 K magni ica ion).
DLS analysis showed a unimodal pa icle size dis ibu ion, wi h a mean hyd ody-
namic diame e o 70
±
9 nm and a PDI o 0.2, while TEM mic og aphs e ealed ha
he HNPs we e cha ac e ized by sphe ical shape and a mean diame e alue close o ha
eco ded by DLS (Figu e 4b). The size o he HNPs desc ibed in his wo k is signi ican ly
smalle compa ed o p e iously epo ed HSA nanopa icles p epa ed by he desol a ion
me hod [
43
–
45
] o sel -assembling p ocesses [
46
–
48
]. Pa icle size is he esul o he combi-
na ion o di e en pa ame e s, including physic-chemical p ope ies o he amphiphile,
p epa a ion me hod, and expe imen al condi ions, ha s ongly a ec he ea angemen o
he polyme s uc u e du ing he sel -assembling s ep. HNPs dimensions p esen an ideal
size ha is la ge enough o accumula e in umo issues ia enhanced pe meabili y and
e en ion (EPR) e ec . Howe e , since he pa icle size is lowe han 100 nm, i is expec ed
o achie e ex ensi e umo pene a ion [49].
3.3. Des abiliza ion Expe imen s
GSH- esponsi e beha io o HNPs was assessed by means o des abiliza ion ex-
pe imen s pe o med in media mimicking he ex acellula and in acellula edox en-
i onmen s. In pa icula , he a ia ion o he dimensional dis ibu ion o micelle-like
nanopa icles was analyzed by DLS a e 24 h incuba ion in phospha e bu e a physio-
logical pH wi h o wi hou GSH 10 mM. The mean diame e o HNPs emained almos
unchanged in he medium, mimicking he ex acellula en i onmen , while in he p esence
o he ipep ide GSH bo h he mean diame e and he PDI d ama ically inc eased o 130
nm and 0.54, espec i ely. This obse a ion con i ms ha HNPs we e esponsi e o GSH,
which can be explained as a consequence o he educ ion o disul ide bonds, esul ing in
he des abiliza ion o he nanopa icle s uc u e [9,50].
3.4. DOX-Loading and Release Expe imen s
A s imuli- esponsi e d ug deli e y sys em is expec ed o quickly elease i s payload
when a ge ed issues o cells a e eached while emaining s able in he sys emic ci cu-
la ion. In his wo k, DOX hyd ochlo ide, employed as a model d ug, was loaded in he
nanopa icles in a one-s ep p ocedu e, a oiding he need o o ganic sol en s o H
2
O
2
o
solubilize he componen s o o ini ia e he molecula assembly o he d ug-nanopa icle
complexes [
43
,
51
,
52
]. The d ug was added a a concen a ion o 32 mg pe g o conju-
ga e du ing he sel -assembling s ep, exploi ing he s ong a ini y be ween DOX and
HSA [
22
,
53
,
54
], and he esul ing DOX@HNPs dispe sion was used as such in he elease
expe imen s assuming ha he o al amoun o added DOX was abso bed by he nanopa i-
cles. This assump ion is suppo ed by li e a u e da a epo ing he ange o DOX:HSA a io
whe e he dissol ed p o ein can comple ely abso b he d ug om a wa e solu ion [46].
Release expe imen s om DOX@HNPs we e pe o med in media mimicking he
physiological condi ions (phospha e bu e a pH 7.4) and he in acellula space (GSH
10 mM) [
55
,
56
]. A con olled elease p o ile was eco ded a pH 7.4 simula ing no mal
Pha maceu ics 2021,13, 304 9 o 16
physiological condi ions: a e a sligh bu s elease a e he i s 30 min (15%), he DOX
cumula i e pe cen age in he eleasing medium does no exceed 26%, indica ing ha he
DOX@HNPs a e s able in he ex acellula medium o heal hy issues and only a small
amoun o d ug would be leaked du ing blood ci cula ion. These esul s e lec he high-
a ini y be ween DOX and HSA in aqueous media a pH alues close o physiological
condi ions. In ac , ecen s udies ha e shown ha he hyd ophilic and hyd ophobic
in e ac ions be ween DOX and albumin esul in he o ma ion o mo e s able complexes
in HSA han o he albumins, such as BSA [21].
The inc ease in he GSH concen a ion up o 10 mM led o a as e elease p o ile,
wi h he elease pe cen age inc easing up o 42% in he i s 30 min and aising 74% a e
24 h
(Figu e 5). This di e en beha io , in acco dance wi h he da a o he des abiliza ion
s udies, is asc ibable o he b eakage o he disul ide linkage. P olonging he elease ime
un il 48 h, no signi ican enhancemen o he elease pe cen age was obse ed, p obably
o he s ong d ug o polyme in e ac ion.
Pha maceu ics 2021, 13, x 9 o 17
can be explained as a consequence o he educ ion o disul ide bonds, esul ing in he
des abiliza ion o he nanopa icle s uc u e [9,50].
3.4. DOX-Loading and Release Expe imen s
A s imuli- esponsi e d ug deli e y sys em is expec ed o quickly elease i s payload
when a ge ed issues o cells a e eached while emaining s able in he sys emic ci cula-
ion. In his wo k, DOX hyd ochlo ide, employed as a model d ug, was loaded in he na-
nopa icles in a one-s ep p ocedu e, a oiding he need o o ganic sol en s o H2O2 o
solubilize he componen s o o ini ia e he molecula assembly o he d ug-nanopa icle
complexes [43,51,52]. The d ug was added a a concen a ion o 32 mg pe g o conjuga e
du ing he sel -assembling s ep, exploi ing he s ong a ini y be ween DOX and HSA
[22,53,54], and he esul ing DOX@HNPs dispe sion was used as such in he elease ex-
pe imen s assuming ha he o al amoun o added DOX was abso bed by he nanopa i-
cles. This assump ion is suppo ed by li e a u e da a epo ing he ange o DOX:HSA
a io whe e he dissol ed p o ein can comple ely abso b he d ug om a wa e solu ion
[46].
Release expe imen s om DOX@HNPs we e pe o med in media mimicking he
physiological condi ions (phospha e bu e a pH 7.4) and he in acellula space (GSH 10
mM) [55,56]. A con olled elease p o ile was eco ded a pH 7.4 simula ing no mal phys-
iological condi ions: a e a sligh bu s elease a e he i s 30 min (15%), he DOX cu-
mula i e pe cen age in he eleasing medium does no exceed 26%, indica ing ha he
DOX@HNPs a e s able in he ex acellula medium o heal hy issues and only a small
amoun o d ug would be leaked du ing blood ci cula ion. These esul s e lec he high-
a ini y be ween DOX and HSA in aqueous media a pH alues close o physiological
condi ions. In ac , ecen s udies ha e shown ha he hyd ophilic and hyd ophobic in-
e ac ions be ween DOX and albumin esul in he o ma ion o mo e s able complexes in
HSA han o he albumins, such as BSA [21].
The inc ease in he GSH concen a ion up o 10 mM led o a as e elease p o ile,
wi h he elease pe cen age inc easing up o 42% in he i s 30 min and aising 74% a e
24 h (Figu e 5). This di e en beha io , in acco dance wi h he da a o he des abiliza ion
s udies, is asc ibable o he b eakage o he disul ide linkage. P olonging he elease ime
un il 48 h, no signi ican enhancemen o he elease pe cen age was obse ed, p obably
o he s ong d ug o polyme in e ac ion.
Figu e 5. D ug elease p o iles o doxo ubicin hyd ochlo ide (DOX) om DOX@HNPs in phos-
pha e bu e a pH 7.4 wi hou (dashed line) and wi h glu a hione (GSH) 10 mM (solid line) a 37
°C.
Figu e 5.
D ug elease p o iles o doxo ubicin hyd ochlo ide (DOX) om DOX@HNPs in phospha e
bu e a pH 7.4 wi hou (dashed line) and wi h glu a hione (GSH) 10 mM (solid line) a 37 ◦C.
3.5. Cy o oxici y S udies
The inhibi ion o cellula p oli e a ion o DOX, HNPs and DOX@HNPs in a cance
cell line (MDA-MB231; human b eas adenoca cinoma) and a no mal cell line (BALB/3T3;
mouse ib oblas s) was e alua ed a e 24 and 48 h using a CCK-8 assay. F om hese
esul s, hal -maximal inhibi o y concen a ion (IC50) alues o ee d ug and nanopa icle
concen a ion we e also calcula ed. Bo h cell lines showed a p og essi e dec ease in cell
su i al in esponse o inc easing concen a ions o d ugs and nanopa icles (Figu e 6). Fo
BALB/3T3 cells, IC
50
alues o DOX a 24 and 48 h we e 1.25 and 1.5
µ
g mL
−1
, espec i ely.
MDA-MB231 cells showed a signi ican ly highe IC
50
a 24 h (>10
µ
g mL
−1
), while a
48 h
, IC
50
was 0.62
µ
g mL
−1
. The high IC
50
alue o DOX a 24 h can be explained due o
he d ug esis ance o he MDA cell line and he majo ole o e lux pumps, including P-
glycop o ein [
57
]. O e all, BALB cells we e signi ican ly mo e sensi i e o DOX compa ed
o MDA cells. Simila ly, he concen a ion o HNPs signi ican ly a ec ed he iabili y o
bo h cance and ib oblas cell lines. The IC
50
o HNPs a e 24 and 48 h incuba ion was
>1 mg mL
−1
, excep o he MDA cells a 48 h ha showed an IC
50 ≈
1 mg mL
−1
. This
inding sugges ed ha HNPs could be easily up ake by MDA-MB231 cells due o he highe
a ini y o CD44 ecep o s p esen on he su ace o he cells and esul ing in a highe
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26
]. The p oli e a ion inhibi ion
s udies o DOX@HNPs we e ca ied ou using an HNPs concen a ion o 0.1 mg mL
−1
o
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