1
Syn hesis and cha ac e iza ion o hyalu onic acid coa ed manganese dioxide
mic opa icles ha ac as ROS sca enge s
Joëlle Bizeaua,b,†, Ch is os Tapeinosa,†, Claudio Ma ellaa, Ai o La añagac, Abhay Pandi a,*
aCen e o Resea ch in Medical De ices (CÚRAM), Na ional Uni e si y o I eland Galway, Galway,
I eland.
bNa ional Poly echnic Ins i u e o Chemical Enginee ing and Technology (ENSIACET), 4 allée Emile
Monso, Toulouse, F ance
cDepa men o Mining-Me allu gy Enginee ing and Ma e ials Science & POLYMAT, Uni e si y o he
Basque Coun y, Bilbao, Spain
†These au ho s con ibu ed equally o his wo k
*Co esponding au ho
joelle.bizeau@ensiace . , c [email protected], ai o .[email p o ec ed], c.ma [email protected],
abhay.pandi @nuigalway.ie
G aphical Abs ac
Highligh s
Syn hesis o hyalu onic acid-coa ed manganese dioxide mic osphe es (HA-Mn-SM) wi h ROS
sca enging abili y
The HA-Mn-SM sca enge H2O2 and gene a e O2 in a con olled manne
The HA-Mn-SM can be loaded wi h an i-oxidan subs ances and hei elease is dependen on lac ic
acid conce a ion
The HA-coa ed mic osphe es a e no cy o oxic
The de eloped pla o m can be used as a DDS o he a ge ed deli e y o an i-oxidan s in
a he oscle o ic issues
Abs ac
A he oscle osis is a ch onic in lamma o y disease o he a e ial wall ha leads o ca dio ascula
diseases which a e he majo cause o dea hs wo ldwide. The e is cu en ly no ea men ha can s op
o e e se he disease. Howe e , he use o mic opa icles wi h an i-in lamma o y p ope ies could
ep esen a p omising ea men . He ein, sphe ical mic opa icles wi h a co e-shell s uc u e and an
a e age diame e o 1 μm we e syn hesized. The mic opa icles we e comp ised o a MnCO3 and MnO2
co e and a 4-a m PEG-amine c oss-linked shell o hyalu onic acid. The HA-Mn-SM mic opa icles we e
loaded wi h D-α- ocophe ol ( i amin-E) (TOC), o ab ica e a a ge ed biocompa ible deli e y pla o m
o he ea men o a he oscle o ic in lamed cells. Loading and elease s udies o TOC demons a ed a
lac ic acid concen a ion dependan con olled elease p o ile o he HA-Mn-SM mimicking he
a he oscle o ic en i onmen whe e lac ic acid is o e -p oduced. The mic opa icles exhibi ed a high
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Colloids and Su aces B: Bioin e aces 159 :
30-38 (2017), which has been published in inal o m a h ps://doi.o g/10.1016/j.colsu b.2017.07.081. © 2017 Else ie
unde CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
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sca enging abili y owa ds H2O2 in addi ion o he con olled gene a ion o O2. The op imal esul s we e
ob ained o 250 μg/mL mic opa icles which in he p esence o 1000 μM H2O2 esul ed in he
sca enging o almos all he H2O2. Ou esul s demons a e ha 50 μg/mL o mic opa icles sca enged
con inuously p oduced H2O2 up o a concen a ion o 1000 μM, a cha ac e is ic ha demons a es he
sus ained he apeu ic e ec o he HA-Mn-SM mic opa icles in an en i onmen ha mimics ha o
in lamed issues. Ou esul s indica e he po en ial use o HA-Mn-SM as a no el pla o m o he
ea men o a he oscle osis. In i o s udies con i med ha he mic opa icles a e no cy o oxic a
concen a ions up o 250 μg/mL and o 72 hou s. These p elimina y esul s indica e he po en ial use o
HA-Mn-SM as a no el d ug deli e y sys em o a he oscle o ic issues.
Keywo ds: A he oscle osis; d ug deli e y; manganese dioxide; mic osphe es; ROS sca enging
Numbe o wo ds: 6876
Numbe o Tables: 0
Numbe o Figu es: 7
3
1.In oduc ion
A he oscle osis is a ch onic in lamma o y disease o he a e ial wall ha damages he endo helium
ha in u n ini ia es an in lamma o y cascade which esul s in he accumula ion o low-densi y
lipop o eins (LDL) known as “bad” choles e ol. In lamma ion leads o he o e p oduc ion o Reac i e
Oxygen Species (ROS) esul ing in he oxida ion o LDL. Oxidized LDL (OxLDL) is up aken by
mac ophages which accumula e in a he oscle o ic issues which along wi h o he mechanisms [1, 2]
con ibu e o he o ma ion o a he oscle o ic plaques which in u n esul s in he onse o ca dio ascula
diseases including co ona y a e y disease, ce eb o ascula disease and pe iphe al a e y disease [3, 4].
Ca dio ascula diseases accoun o he highes pe cen ages o mo bidi y and mo ali y wo ldwide. In
he Eu opean Union 42% o all dea hs in 2012 esul ed om a disease o he ci cula o y sys em, while
cance (malignan neoplasms), he second mos p e alen cause o dea h, accoun ed o only 24.8% o
dea hs. [5]. In he Uni ed S a es in 2013, one o e e y h ee dea hs was caused by a ca dio ascula
disease. E e y 34 seconds, someone su e ed a co ona y e en and e e y 84 seconds, someone died om
a co ona y e en [6]. To da e, he ea men s o a he oscle osis comp ise mos ly o p e en i e s a egies
ha ac on a pe son’s li es yle by educing a ious isk ac o s including smoking and by p omo ing
egula exe cise no ably o con ol he pe son’s weigh [3, 4, 7, 8]. Un o una ely, hese ac ions canno
s op he p og ession o e e se he o ma ion o a he oscle o ic plaques. Ano he app oach o he
ea men o a he oscle osis includes he use o d ugs such as a) s a ins ha educe LDL and inc ease
high-densi y lipop o eins (HDL) [9, 10], b) an i-pla ele medica ion [4, 8] and c) be a blocke medica ion
[4, 8] ha educe hea a e and blood p essu e. When hese ea men s ail o mee he equi ed
s anda ds, mo e agg essi e ea men s including angioplas y and s en placemen o enda e ec omy can
be applied. As men ioned be o e, one o he mechanisms ha lead o a he oscle osis is he
o e p oduc ion o ROS and he ans o ma ion o LDL o OxLDL. Al hough he abo e-men ioned
ea men s can delay he p og ession o a he oscle osis, hey do no lead o i s eg ession and one
possible eason o his may be ha hey do no ake in o conside a ion he in lamma o y en i onmen
o he disease.
Manganese dioxide (MnO2) is a well-known ca alys o he decomposi ion o hyd ogen pe oxide
(H2O2 o wa e and oxygen. A numbe o s udies ha e a emp ed o unde s and he mechanism o ac ion
[11] and o e alua e i s e icacy as a ca alys in ela ion o he pa icles’ size, shape and composi ion
[12-15], and he a e o decomposi ion.
I has also been shown ha MnO2 nanopa icles eac di ec ly wi h H2O2 o p oduce wa e , oxygen and
Mn2+ ions [16], and Mn2+ ions a e ound inside nume ous biological sys ems [17]. In addi ion, i has
been demons a ed ha Mn2+ ions p oduce a p o ec i e e ec agains H2O2-media ed inju ies in ce ain
condi ions [18].
To da e, se e al s imuli- esponsi e d ug deli e y sys ems o he ea men o cance ha e been
de eloped [19-21] and e en hough he ca aly ic and eac i e p ope ies o MnO2 mic o- and nano-
pa icles ha e been used la gely o he de elopmen o biosenso s and supe capaci o s [13, 22], hei
ole as a d ug deli e y sys em (DDS) is jus beginning o be in es iga ed [22-26]. Liu e al de eloped a
mic ochip in which mic ochannels a e composed o a luid laye o polydime hylsiloxane and a
se pen ine MnO2 nano ibe subs a e wi h an epi helial cell adhesion an ibody coa ing. These mic ochips
cap u e and elease ci cula ing umo cells in o de o measu e and analyze hem o diagnos ic pu poses
[27]. In addi ion, manganese-based sys ems including MnO2 nanopa icles ha e been used as imaging
agen s in a ious diseases [28-32].
Hyalu onic acid (HA) is a ca bohyd a e composed o D-Glucu onic acid and N-ace yl-D-glucosamine
linked by al e na ing β-1, 4 and β-1, 3 glycosidic bonds which is ound in he ex acellula and
pe icellula ma ix [33] as also inside cells. I s abundan p esence in he body ensu es he
biocompa ibili y o his na u al polyme , and i s use as a coa ing ma e ial p o ides p o ec ion o he
a ious mic o- and nano- cons uc s om he immune sys em. I s main ecep o , CD44 is no only
o e exp essed in a ious cance umou cells bu also in in lamma o y cells [34] including ac i a ed
mac ophages which play an impo an ole in he de elopmen o a he oscle o ic plaques [35]. In
addi ion, S abilin-2, ano he HA ecep o , is highly exp essed in a he oscle o ic plaques [35]. The
4
biocompa ibili y and biodeg adabili y o HA, in addi ion o he he p esence o i s ecep o in cance ous
and in lamed issues, ende s i a sui able ma e ial o coa ing and a ge ed ea men applica ions. Fo
hese easons, HA is used o he syn hesis o HA nanopa icles [35] as well as a ma e ial o coa ing
d ug deli e y sys ems including chi osan [36], liposomes [37] and nanos uc u ed lipid ca ie s [38].
E en hough he exac mechanism o ac ion o i amin-E as an an i-oxidan is no well unde s ood, i is
belie ed ha i p o ec s LDL om ROS oxida ion [39] and ha i modula es a a ie y o in lamma o y
p ocesses [40] in ol ed in a he ogenesis. Fu he mo e, D-α- ocophe ol (TOC), he mos ac i e o m o
i amin-E, inhibi s smoo h muscle cell p oli e a ion and down egula es monocy e ec ui men and
sca enge ecep o CD36 exp ession, educing he up ake o LDL by mac ophages [41]. I also ac s as
an inhibi o o p oin lamma o y cy okines p oduced by endo helial and immune cells, and as a
supp esso o he exp ession o adhesion molecules o endo helial cells. I also ac s as a educ o o he
a ac ion o monocy es o in lamma o y si es a he a e ial wall [39].
Taking in o conside a ion he p ope ies o all he abo e-men ioned ma e ials, we syn hesized and
cha ac e ized a mul i unc ional d ug deli e y sys em ha can be used as a po en ial ea men o
a he oscle o ic issues. In his s udy, we p esen he syn hesis o a new d ug deli e y sys em consis ing
o MnO2 sphe ical mic opa icles coa ed wi h HA. This pla o m can ac as a ROS sca enge due o he
inco po a ion o MnO2 sphe ical mic opa icles, and also p o ides a con olled elease p o ile o an
encapsula ed d ug ollowing he dissolu ion o he mic opa icles in lac ic acid, a subs ance ha is
o e p oduced in a he oscle o ic issues. We hypo hesized ha he combina ion o MnO2 mic opa icles
and D-α- ocophe ol would inhibi he p og ession o a he oscle osis by educing he excess o ROS and
by inhibi ing he oxida ion o LDL and he subsequen up ake o oxidized LDL by he mac ophages.
2. Ma e ials and me hods
2.1. Ma e ials
Manganese (II) ni a e e ahyd a e (Mn(NO3)2.4H2O), sodium ca bona e (Na2CO3), po assium
pe mangana e (KMnO4), hyd ogen pe oxide (H2O2) and 2-(N-mo pholino) e hane sul onic acid (MES
C6H13NO4S) we e used as ecei ed om Sigma-Ald ich. L-(+)-Lac ic acid (C3H6O3), sodium chlo ide
NaCl and D-α-Tocophe ol polye hylene glycol 1000 succina e (TOC) we e used as ecei ed om Sigma
Li e Sciences. N-(3-dime hylaminop opyl)-N’-e hyl ca bodiimide hyd ochlo ide (EDC) and N-
hyd oxysuccinimide (NHS) we e used as ecei ed om Fluka Analy ical. 4-a m PEG
amine(pen ae y h i ol)•HCl was used as ecei ed om JenKem Technology. A 10mg/mL hyalu onic
acid solu ion was p epa ed by dissol ing sodium hyalu ona e (800kDa - 1.2MDa) ecei ed om
Li eco e Biomedical in deionized wa e and s o ed a 4°C. E hanol (C2H5OH) was used as ecei ed om
Lennox.
Fo he in i o s udies, 3T3 ib oblas ic cells om a s we e used. Hank’s Balanced Sal Solu ion
(HBSS) and Dulbecco’s Modi ied Eagle’s Medium (DMEM) wi h and wi hou Phenol Red we e
pu chased om Sigma Li e Sciences. T ypsin-EDTA was used as ecei ed om Sigma-Ald ich and
Alama Blue® was used as ecei ed om Li e Technologies/Biosciences.
2.2. Cha ac e iza ion me hods
The chemical composi ion o he sphe es was e i ied using a Fou ie T ans o m In a ed
spec ome e Va ian 660-IR. The ans o ma ion o he KMnO4 in o MnO2 was s udied using UV-Vis
spec opho ome y (Va ioskan Flash Spec al Scanning Mul imode Reade wi h Skani so wa e 2.4.3
om The mo Scien i ic) and X-Ray powde di ac ion analysis (Inel Equinox 3000 X- ay powde
di ac ome e ). The mo phological cha ac e iza ion o he mic opa icles was ca ied ou using a
Scanning Elec on Mic oscope (SEM) wi h EDX Analysis Sys em, a a wo king ol age and wo king
cu en o 5 kV and 10 mA espec i ely. ζ-po en ial was de e mined using Dynamic Ligh Sca e ing
(Ze asize Nano Se ies, Mal e n Ins umen ). A Va ioscan Flash UV-Vis pla e eade was used o
eading luo escence o assess he H2O2 sca enging e ec o he mic opa icles and he eadou s we e
5
measu ed a λex=540 nm/ λem=590 nm. Oxygen gene a ion was measu ed using a Mic ox 4 ace ib e
op ic oxygen me e ® wi h a PS 7-O2 mic o-senso . Da a was collec ed e e y wo seconds o 60 minu es
using he Da a Manage so wa e o P esens® and analyzed using O iginP o®.
Quan i ica ion o he amoun o loaded TOC was ca ied ou using Nuclea Magne ic Resonance (JEOL-
400MHz NMR Ins umen ) o he hyd ogen nucleus. Du ing he washing o he sphe es, he i s
supe na an was collec ed and ozen a -80 °C o 30 minu es be o e being ans e ed o he eeze
d ye . Then, he sample was dissol ed in deu e a ed DMSO. Fou samples con aining 0.5, 1.0, 1.5 and
2.0 mg/mL o TOC in deionized wa e we e p epa ed as desc ibed o ob ain he cha ac e is ic spec um
o he TOC and o plo a calib a ion cu e using he in eg a ion o he peaks.
2.3. Syn hesis o MnO2 sphe ical mic opa icles (Mn-SM)
2.3.1. P epa a ion o MnCO3 empla e
The MnCO3 mic opa icles we e syn hesized acco ding o a modi ied p o ocol p e iously epo ed
[42]. B ie ly, 20 mL o a 0.1 M Mn(NO3)2·4H2O solu ion was added o 20 mL o a 0.1 M Na2CO3
solu ion wi h a low a e o 10 mL/min unde igo ous s i ing. The solu ion u ned milky whi e om
he addi ion o he i s d op sugges ing he ins an o ma ion o manganese ca bona e. A e he addi ion
o Mn(NO3)2, he s i ing was s opped and he sphe es we e le o 15 minu es a oom empe a u e
be o e being washed h ee imes wi h deionized wa e . The p oduc was collec ed using cen i uga ion
a 8000 pm o 5 minu es. A e he washings, he pa icles we e d ied and s o ed a oom empe a u e.
2.3.2. P epa a ion o he MnO2 mic opa icles
The MnO2 mic opa icles we e syn hesized acco ding o a modi ied p o ocol p e iously epo ed
[43]. A mola a io o KMnO4 o MnCO3) o 1 o 50 was used o he syn hesis. 10 mL o he KMnO4
solu ion was added o he MnCO3 mic opa icles and he solu ion was s i ed igo ously o 30 minu es.
The solu ion u ned b own and hen he sphe es we e collec ed using cen i uga ion and washed ou
imes wi h deionized wa e . The cen i uga ion used o his s ep was 8000 pm o 5 minu es. A e he
washings, he pa icles we e d ied and s o ed a oom empe a u e.
2.4. Coa ing o Mn-SM wi h Hyalu onic Acid (HA-Mn-SM)
30 mg o Mn-SM was suspended in 1.50 mL o a 0.15 M NaCl solu ion, (pH 6.5) unde igo ous
s i ing. Then 0.5 mL o a 10 mg/mL HA solu ion was added. The solu ion was s i ed o 30 minu es
be o e being cen i uged a 4500 pm o one minu e o sepa a e he pa icles om he liquid. The
pa icles we e washed h ee imes wi h a 0.01 M NaCl solu ion (pH 6.5) be o e being d ied and s o ed
a oom empe a u e.
2.5. C oss-linking wi h 4-a m-PEG-amine (4S-PEG)
30 mg o HA-Mn-SM mic opa icles, 15 mg o EDH and 15 mg o NHS we e suspended in 4 mL o
MES bu e a pH 6 and s i ed o 30 minu es. Then he pH was inc eased wi h a 10 M NaOH solu ion
o pH 7.4 be o e he addi ion o 5 mg o 4-a m PEG-amine dissol ed in 1 mL o deionized wa e . The
solu ion was s i ed o 5 hou s be o e being cen i uged a 8000 pm o 5 minu es and washed i e
imes wi h deionized wa e . The sample was hen d ied and s o ed a oom empe a u e.
2.6. E ec o lac ic acid in he HA-Mn-SM mic opa icles
The e ec o lac ic acid was es ed on plain MnCO3 mic opa icles and on HA-Mn-SM mic opa icles
c oss-linked wi h 4S-PEG. 10 mg o MnCO3 mic opa icles was placed in 5 mL o lac ic acid 0.01 M
(pH 3) while s i ing. E e y 5 minu es he solu ion was changed wi h esh lac ic acid o he same
concen a ion added in o de o main ain a s able pH. This was done o simula e he con inuous
gene a ion o lac ic acid in he body. The same p ocedu e was ollowed o he c oss-linked HA-Mn-
SM.
6
2.7. Loading and Release s udies using D-α- ocophe ol
2.7.1. Loading by en apmen du ing he syn hesis o MnCO3 mic opa icles
The loading o TOC was ca ied ou du ing he syn hesis o MnCO3 mic opa icles. 6 mg o TOC
was dissol ed in 1 mL o deionized wa e and 1 mL o a 0.1 M Na2CO3 solu ion was added unde
igo ous s i ing. Then, 1 mL o a 0.1 M Mn(NO3)2·4H2O solu ion was added d op by d op wi h a
sy inge while s i ing. Following his addi ion, he solu ion was milky whi e indica ing ha MnCO3 had
been syn hesized. The s i ing was s opped and he mic opa icles we e le a oom empe a u e o 15
minu es be o e being washed h ee imes wi h deionized wa e . The i s supe na an was kep o NMR
analysis.
2.7.2. Loading by di usion in HA-Mn-SM
30 mg o he HA-Mn-SM and 10 mg o TOC we e suspended in 5 mL o deionized wa e and s i ed
o 24 hou s. A e 24 hou s, he mic opa icles we e washed h ee imes wi h deionized wa e . The
cen i uga ion used o his s ep was 4500 pm o one minu e. The supe na an s be o e and a e he
i s washing we e kep o NMR analysis.
2.7.3. Release s udies o HA-Mn-SM
Fo he elease s udies he same p ocedu e ha was used o s udy he e ec o lac ic acid on he HA-
Mn-SM mic opa icles was ollowed. Lac ic acid was changed e e y i e minu es and he supe na an
was collec ed, eeze-d ied and esuspended in DMSO. The amoun o eleased TOC was analyzed using
NMR.
2.8. ROS sca enging e ec o he HA-Mn-SM
A luo ome ic Hyd ogen Pe oxide Assay Ki was used o assess he sca enging e ec o he
mic opa icles’ owa ds H2O2. The concen a ions es ed we e 50, 100 and 250 μg/mL o he
mic opa icles and 50, 100 and 1000 μM o H2O2. The calib a ion cu e was de eloped o 0, 2.5, 5
and 10 µM o H2O2.
Th ee di e en solu ions o he c oss-linked HA-Mn-SM 100, 200 and 500 μg/mL and h ee di e en
solu ions o H2O2 100, 200 and 1000 μM we e p epa ed in deionized wa e and mixed in a 1:1 a io in a
inal olume o 1 mL. The solu ions we e shaken o one hou . Then, 50 μL o each solu ion was added
in a 96-well pla e duplica e. 50 μL o he bu e was added o each well. This bu e was p epa ed as pe
he manu ac u e ’s p o ocol. The 96-well pla e was co e ed wi h oil and shaken o a pe iod o wen y
minu es be o e luo escence alues we e measu ed.
2.9. Oxygen gene a ion
0.5 mL o a ious concen a ions o he HA-Mn-SM mic opa icles solu ion (50, 100 and 250 µg/mL)
we e in oduced in o a ial closed wi h a sep um, using a sy inge. While s i ing he oxygen, he senso
was imme sed in he solu ion and le o i e minu es o equilib a e he oxygen concen a ion. A e
i e minu es, 0.5 mL o di e en concen a ions o H2O2 solu ion (50, 100 and 1000 µM) we e added.
The measu emen s we e ake o e a pe iod o one hou .
2.10. In i o s udies
Fo he in i o s udies, he Alama Blue® assay was used o check he me abolic ac i i y and he
subsequen cy o oxici y o he mic opa icles on 3T3 a ib oblas s.
2.10.1. Cell seeding
The cells we e cul u ed in a T75 lask a 37 °C and 5% CO2 un il he popula ion con luency eached
80%. Be o e seeding he cells in o he well pla es, he lask was aken ou om he incuba o and he
cells we e checked unde he mic oscope o g ow h and any sign o con amina ion. The old medium
was disca ded and eplaced by 10 mL o HBSS o inse cells and emo e any aces o medium. The
7
lask was shaken gen ly and HBSS was disca ded. To de ach he cells, 3 mL o T ypsin-EDTA was
added in o he lask which was hen incuba ed o one minu e a 37 °C wi h 5% o CO2. The lask was
shaken and gen ly apped o help de ach he cells, and checked unde he mic oscope. 7 mL o DMEM
was added o deac i a e ypsin and hen he cells we e cen i uged o i e minu es a 1200 pm. The
supe na an was disca ded and he cells we e dispe sed in 10 mL o esh DMEM. 20 µL was collec ed
and placed on he hemocy ome e o coun cells. The app op ia e olume was aken o ill wel e wells
wi h 500 µL o he solu ion a a inal cell concen a ion o 78,125 cells/cm2. The pla es we e hen placed
in he incuba o a 37 °C wi h 5% o CO2 o 24 hou s.
2.10.2. Incuba ion o he cells wi h he mic opa icles
The mic opa icles we e s e ilized by esuspension in 70% e hanol o e nigh , and be o e use, hey
we e washed wice wi h HBSS and once wi h DMEM. Then he mic opa icles we e dispe sed in
DMEM a concen a ions o 50, 100 and 250 µg/mL. The pla es we e aken ou om he incuba o and
he cells we e checked unde he mic oscope o g ow h and any sign o con amina ion. The media was
hen emo ed and 500 µL o HBSS was added o each well o wash he cells. HBSS was hen emo ed
and 500 µL o he desi ed mic opa icle solu ions we e added and placed back in he incuba o a 37 °C
wi h 5% CO2. The me abolic ac i i y o he cells was s udied a e 24 hou s and 72 hou s.
2.10.3. Alama Blue® assay
The pla e was aken ou om he incuba o and he media was emo ed. The cells we e washed once
wi h HBSS. Then, 200 µL o an Alama Blue® solu ion, 10% in DMEM, wi hou Phenol Red, was added
o each well. Con ol samples in wells no seeded wi h cells wi h Alama Blue® only and DMEM wi hou
phenol ed only we e also s udied. The pla e was p o ec ed om ligh using oil and incuba ed a 37 °C
wi h 5% CO2 o 4 hou s. Then, he solu ions om he 48-well pla es we e ans e ed o a 96-well pla e,
100 µL in each well, and he abso bance was ead a 570 nm and 600 nm. The pe cen age o Alama Blue
educ ion was calcula ed using he ollowing equa ions:
%Alama Blue educ ion = (ALW – (AHW×R0))×100 (Eq. 1)
Whe e ALW is he abso bance a a low wa eleng h, AHW is he abso bance a a high wa eleng h and R0
is he co ela ion ac o .
R0 = AOLW/AOHW (Eq. 2)
8
3. Resul s and discussion
3.1. Syn hesis o MnO2 sphe ical mic opa icles Mn-SM
The p o ocol used in his s udy o he ab ica ion o he sphe ical mic opa icles was a combina ion
o wo di e en p ocedu es. We syn hesized he MnCO3 co e syn hesized using h he p o ocol epo ed
by Pou mo aza i e al. [42] and he condi ions epo ed by Fei e al. [43] o ob ain MnO2 by eac ion o
MnCO3 wi h KMnO4. The i s e idence o he o ma ion o MnO2 mic opa icles was he change o
colou om whi e (MnCO3) o da k b own (MnO2). Fo ma ion o MnO2 mic opa icles was also
suppo ed by he FT-IR spec um (Figu e 1a). Mo e speci ically, he h ee cha ac e is ic peaks o
MnCO3 a 722 cm-1, 861 cm-1 and 1392 cm-1 co espond o he C-O bond. In Figu e 1b, hese peaks can
also be obse ed bu he ou h peak a 472 cm-1 ha co esponds o he O-Mn-O bond o MnO2 is also
e iden . The SEM pic u es o he MnCO3 (Figu e S1a - see supplemen a y in o ma ion) show ha he
pa icles a e sphe ical wi h a diame e ha anges om 0.85 µm o 1.13 µm. Figu e S1b (see
supplemen a y in o ma ion) shows ha he ans o ma ion o MnCO3 o MnO2 does no a ec he shape
o he size o he pa icles bu changes he su ace mo phology. Addi ional e idence o he p esence o
MnO2 was he educ ion o he ζ-po en ial om +16.17 ± 6.42 mV o +10.37 ± 5.80 mV (Table S1) (see
Supplemen a y in o ma ion).
I is known ha MnO2 eac s wi h H2O2 esul ing in he syn hesis o H2O, O2, and Mn2+ in acco dance
wi h he ollowing equa ion 3:
MnO2 + H2O2 + 2H+ → 2H2O + O2 + Mn2+ (Eq. 3)
3.2. Coa ing wi h hyalu onic acid and c oss-linking using 4-a m-PEG-amine (4S-PEG)
Figu e 1c shows he HA-coa ed mic opa icles ob ained wi h he second mix u e. The su ace
mo phology changed wi h he appea ance o a needle-like ma e ial a ound he pa icles. Fu he
e idence sugges ing a success ul coa ing was he su ace cha ge (ζ-po en ial) alue o ou ma e ial. Ou
esul s (Table S1-see supplemen a y in o ma ion) show ha he posi i e cha ge o he ma e ial was
con e ed in o a nega i e cha ge, om +10.37 ± 5.80 mV o -37.97 ± 6.21 mV. The change o sign in
he ζ-po en ial can be a ibu ed o he dep o ona ion o he ca boxylic g oups con ained in each
disaccha ide o HA [33]. Knowing ha HA has a pKa o a ound 4 because o hese ca boxylic g oups
and ha he measu emen was ca ied ou a pH 6.5 o he solu ion used in he coa ing p ocedu e and
5.5 o he solu ion used o he measu emen o ζ-po en ial, i is sa e o assume ha comple e
dep o ona ion o he unc ional g oup in hese condi ions has aken place, p o iding a nega i e cha ge
o HA. The change o ζ-po en ial du ing he coa ing p ocedu e sugges s ha HA is bonded o he pa icles
by elec os a ic in e ac ions, and hese esul s a e consis en wi h he esul s ob ained by Ra a e al [37].
Ano he expe imen was ca ied ou using 1.80 mL o he NaCl solu ion and 0.20 mL o HA solu ion.
As hese condi ions and esul s a e simila o hose al eady p esen ed in his pa ag aph, hey a e no
p esen ed again he e.
Du ing he op imiza ion o he c oss-linking p ocedu e, wo di e en bu e s (PBS(1X) and MES
bu e ), as well as wo di e en concen a ions o 4S-PEG, 2.5 mg, and 5 mg, we e used. The p ocedu e
ha used PBS(1X) has been excluded due o he p esence o la ge amoun s o sal s a ound he pa icles
ha could no be comple ely emo ed e en a e nume ous washings. As can be obse ed in Figu e 1d,
when he highe amoun o he c osslinke and he MES bu e was used, he mo phology o he su ace
did no change and he sphe es kep hei o iginal size and shape.
3.3. E ec o lac ic acid in he HA-Mn-SM mic opa icles
To de e mine he a e o he MnCO3 componen in he body and mo e speci ically in an
a he oscle o ic o in lamma o y en i onmen , a dissolu ion expe imen was conduc ed using lac ic acid
aiming o s imula e he con inuous and o e p oduc ion o his ype o acid in in lamed cells. A e
changing he sol en ou imes, MnCO3 was comple ely dissol ed so emo al o his componen was
no necessa y du ing he syn hesis o he pla o m.
9
In o de o check how he coa ing a ec s he dissolu ion o he co e and he subsequen elease o he
loaded TOC, a dissolu ion es using lac ic acid was pe o med. Figu e 1e and 1 shows he esul : no
sphe es can be obse ed bu a needle-like ma e ial is p esen . So, all mic opa icles a e des oyed du ing
he dissolu ion es . This can be explained by he ac ha when MnCO3 is dissol ed by lac ic acid, he
esul ing MnO2 sphe es a e po ous and a e no s ong enough o keep hei shape. This was a good esul
o he elease: he dissolu ion o MnCO3 allows a con olled elease o he encapsula ed d ug and hen
MnO2 can sca enge he ROS. Ul ima ely, he sphe es dissocia e and can be easily elimina ed by he
body.
3.4. Loading and Release s udies using wi h D-α- ocophe ol (TOC)
Loading o he HA-Mn-SM was ca ied ou by op imising a ious pa ame e s including he concen a ion
o TOC du ing loading, he loading ime and he loading me hod (loading by di usion and loading by
en apmen du ing he syn hesis o he co e). These pa ame e s we e a ied o de e mine he op imal
loading condi ions o he D-<alpha>- ocophe ol ( i amin-E, TOC). These expe imen s iden i ied he
op imal condi ions o TOC loading o be a TOC concen a ion o 2 mg/mL, a loading ime o 24 hou s,
and loading u ilising he di usion me hod. Loading by en apmen led o e y low encapsula ion
e iciencies and loading u ilising he di usion me hod o mo e han 24 hou s did no signi ican ly
inc ease he encapsula ion e iciency (da a no p esen ed). Lowe concen a ions o TOC esul ed in
lowe loading e iciency which is a ibu ed o he loss o he ma e ial du ing he washing s eps.
Fu he mo e, loading by en apmen du ing he syn hesis o he mic opa icles was less e ec i e han
loading by di usion which was a ibu ed o he high po osi y o he mic opa icles which ende ed hem
unable o en ap he loaded molecules. On he o he hand, coa ing wi h HA inc eased he elec os a ic
in e ac ions and o med a ba ie a ound he mic opa icles which limi ed he capaci y o he loaded
molecules encapsula ed by di usion o be eleased. Figu e 2a ep oduced below illus a es ha he
encapsula ion e iciency o TOC was app oxima ely 60% unde he condi ions desc ibed abo e.
The elease s udies we e conduc ed using lac ic acid a a concen a ion o 10 mM. The mic opa icles
we e ea ed wi h 10 mM lac ic acid and he lac ic acid solu ion was added e e y 5 minu es. The lac ic
acid solu ion was changed in o de o mimic he con inuous p oduc ion o lac ic acid which occu s in
a he oscle o ic issues and o main ain a s able pH ( he pH would change due o he dissolu ion o MnCO3
a e i s eac ion wi h lac ic acid).
The esul s om he elease s udies a e p esen ed in Figu e 2b, which demons a ed ha con olled
elease o TOC occu s in a lac ic acid concen a ion dependen manne . I should be no ed ha he lac ic
acid concen a ion is he cumula i e concen a ion ha esul s om he addi ion o a new lac ic acid
solu ion e e y 5 minu es. The highe elease p o ile obse ed a e 20 minu es and 3 addi ions o he
lac ic acid solu ion (cumula i e 40 mM lac ic acid) was app oxima ely 70%. A elease s udy was also
pe o med using he HA-Mn-SM in he p esence o 10mM lac ic acid o e a pe iod o 24 hou s. The
esul s demons a ed ha he elease o TOC was app oxima ely 10%. This esul demons a ed ha TOC
was no eleased o e ime, bu only in he p esence o a high concen a ion o lac ic acid.
3.5. ROS sca enging e ec on Hyd ogen Pe oxide H2O2
The sca enging abili y o he mic opa icles is p esen ed in Figu e 3. I is impo an o no e ha e en a
he lowes concen a ion o mic opa icles (50 μg/mL), he pa icles educed he concen a ion o H2O2
om 1000 μM o jus 60 μM. When he concen a ion o he mic opa icles was inc eased, he sca enging
e ec was mo e p onounced. The op imal esul s we e ob ained o 250μg/mL mic opa icles which in
he p esence o 1000 μM H2O2 sca enged almos all o he H2O2.
The esul s p esen ed in Figu e 3 demons a e a dose-dependen sca enging e ec since he pe cen age
o H2O2 sca enged is inc eased when he HA-Mn-SM concen a ion is inc eased. In addi ion, gi en ha
H2O2 is con inuously p oduced in in lamed issues, ou esul s demons a e ha 50 μg/mL o
mic opa icles a e capable o sca enging con inuously p oduced H2O2 up o a concen a ion o 1000 μM,
a cha ac e is ic ha demons a es he sus ained he apeu ic e ec o he HA-Mn-SM mic opa icles.
16
lac ic acid (LA). The lac ic acid was changed e e y i e minu es wi h a esh addi ion o lac ic
acid in o de o main ain a pH alue o 3. The concen a ion on he x axis is he cumula i e lac ic
acid concen a ion a e each change. The da a we e p esen ed as he mean ± SD (n=3).
Figu e 3 : Concen a ion o H2O2 (50 uM, 100 uM and 1000uM) a e incuba ion wi h HA-Mn-
SM mic opa icles o 15 minu es a concen a ions o 50, 100 and 250 µg/mL Resul s a e
p esen ed as mean ± SD (n=2)
Figu e 4 : a) and b) The measu ed amoun o gene a ed oxygen as a unc ion o imeusing
a ious amoun s o MnO2 mic opa icles and a ious concen a ions o H2O2. =5 minu es
17
co esponds o he addi ion o H2O2 o he mic opa icles solu ion. Table S2 (see Supplemen a y
in o ma ion) desc ibes in de ail he concen a ions used in his expe imen .
