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EFFECTS OF CHITOSAN/CHONDROITIN SULFATE POLYSACCHARIDE LIDS ON
AMOXICILLIN DRUG RELEASE FROM MICROCONTAINERS
1,2Ilkay KARAKURT, 3Kho shid KAMGUYAN, 3Ca men Milian GUIMERA, 1Kadi OZALTIN,
3Anja BOISEN, 1,4Ma ian LEHOCKY
1Cen e o Polyme Sys ems, Uni e si y Ins i u e, Tomas Ba a Uni e si y in Zlin, Zlin, Czech Republic, EU,
yka aku @u b.cz, [email p o ec ed], lehocky@u b.cz
2Depa men o Heal h Technology, DTU Heal h Tech, Technical Uni e si y o Denma k, Lyngby, Denma k,
EU, [email p o ec ed]k
3The Danish Na ional Resea ch Founda ion and Villum Founda ion’s Cen e o In elligen D ug Deli e y and
Sensing Using Mic ocon aine s and Nanomechanics (IDUN), Technical Uni e si y o Denma k, Lyngby,
Denma k, EU, [email p o ec ed], aboi@d u.dk
4Facul y o Technology, Tomas Ba a Uni e si y in Zlín, Zlín, Czech Republic, EU, lehocky@u b.cz
h ps://doi.o g/10.37904/nanocon.2021.4352
Abs ac
Mic ocon aine s a e mic ome e -sized cylind ical ese oi -based de ices se ing as d ug ca ie s o o al
adminis a ion sys ems wi h ensu ing a high d ug concen a ion a he desi ed si e o abso p ion and p o iding
a sus ained esiden ime. These polyme ic mic o-cylinde s consis o d ug- illed ca i ies and can be
unc ionalized by sealing wi h polyme ic lids o p o ec he ac i e pha maceu ical ing edien agains deg ada ion
be o e eaching he abso p ion si e. The p esen s udy aimed o de elop biopolyme -based lids o
mic ocon aine s using polysaccha ides o he p olonged elease o an an ibio ic. Amoxicillin was chosen as a
model d ug and loaded in o mic ocon aine s using he polydime hylsiloxane masking echnique, ollowed by
sp ay coa ing wi h chi osan (CS) and chond oi in sul a e (ChS) as polyme ic lids on o he ca i y o he de ices.
The s uc u al and physicochemical p ope ies o he mic ocon aine s we e in es iga ed. Mo eo e , he e ec
o polysaccha ide-based lids on he d ug elease beha io was u he analysed and discussed. The hickness
and mo phology o he lids we e cha ac e ized by p o ilome y and SEM be o e and a e sp ay coa ing. The in
i o elease p o iles e ealed a as and an immedia e elease o amoxicillin om only ChS-coa ed
mic ocon aine s as in he uncoa ed ones, and slow, p olonged elease om only CS-coa ed ones. Howe e ,
he mic ocon aine s wi h CS-ChS lids showed a biphasic elease pa e n ha consis s o a lesse bu s elease
in he ea ly s age o ea men , ollowed by a mo e sus ained elease. The e o e, he o ma ion o
polyelec oly es be ween CS and ChS plays an impo an ole in con olling he elease p ocess. The esul s
sugges ha he CS-ChS based lids o mic ocon aine s can be use ul in biodeg adable, ailo -made con olled
d ug deli e y sys ems.
Keywo ds: Mic ode ices, polysaccha ide lids, sp ay coa ing, d ug deli e y, sus ained elease
1. INTRODUCTION
Bac e ial in ec ions ha e been one o he mos p e alen and se ious h ea s o human heal h o decades [1].
Faced wi h such a se e e si ua ion, con olled use o an ibio ics s ill seems o be he mos e ec i e me hod in
in ec ion con ol [2]. Amoxicillin (α-amino-hyd oxybenzylpenicillin), an aminopenicillin an ibio ic wi h b oad-
spec um an ibac e ial ac i i y, is used o ea se e al in ec ions esul ed om mos g am-posi i e and g am-
nega i e bac e ial pa hogens [3]. Howe e , he main limi a ions associa ed wi h his an ibio ic a e possessing
a sho hal -li e and he need o highe doses o e ec i ely show a he apeu ic ac i i y which causes se e e
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side e ec s in u n [4,5]. In o de o a oid o e use o an ibio ics and o imp o e hei pha macokine ic
p ope ies, encapsula ion in o liposomes, emulsions, polyme ic mic o-and nanopa icles a e being ac i ely
de eloped [6].
Recen ly, he ad ances in ese oi -based mic ode ices such as mic ocon aine s held g ea p omises as o al
dosage o ms owa d deli e ing he pha maceu icals o he abso p ion si e, p o iding a unidi ec ional elease,
and imp o ing o al bioa ailabili y [7]. In addi ion, wi h he unc ionaliza ion by polyme ic lids, con olled d ug
elease p o iles and addi ional mucoadhesion can be achie ed [8].
Among na u al polysaccha ides, chi osan and chond oi in sul a e ha e been widely used as elease modi ie s
in d ug deli e y applica ions owing o hei unique p ope ies, including biodeg adabili y, an ibac e ial ac i i y,
and excellen biocompa ibili y [9]. Chi osan ac s as a polyca ion due o he ee NH2 g oups in i s s uc u e,
which in u n eac s wi h nega i ely cha ged subs ances such as chond oi in sul a e, ha consis s o SO42− and
COO− g oups, making i an op imal candida e o o m a polyelec oly e complex (PEC) [10,11].
In his s udy, a BCS class I an ibio ic was loaded in o mic ocon aine s h ough he polydime hylsiloxane
(PDMS) masking echnique and we e coa ed wi h he polysaccha ides by using he Laye -by-laye (LBL)
sp aying echnique. The mic ocon aine s we e unc ionalized by applying wo laye s as lids on op o he
mic ocon aine s, he i s laye was ei he chi osan o chond oi in sul a e. The amoxicillin loaded and
polysaccha ide-coa ed mic ocon aine s we e e alua ed in i o o mo phology and d ug elease p ope ies.
2. MATERIALS AND METHODS
Silicon (Si) wa e s (4-in, b100N n- ype) we e ob ained om Okme ic (Van aa, Finland), and SU-8 2075 and
SU-8 de elope s we e pu chased om Mic o esis Technology GmbH (Be lin, Ge many). Low molecula
weigh chi osan (50-190 kDa) wi h 75-85% deg ee o deace yla ion, chond oi in sul a e A sodium sal (60%
balance is chond oi in sul a e C), ace ic acid (100.5%) and PBS able s we e pu chased om Sigma Ald ich
(P ague, Czech Republic). Sylga d® 184 wo-pa Silicone Elas ome ( iscosi y (mixed) = 3500 cP, speci ic
g a i y (cu ed) = 1.03) was ob ained om Dow Co ning (MI, USA). Amoxicillin ihyd a e was p o ided om
Tokyo Chemical Indus y (Tokyo,Japan) and Milli-Q deionized wa e was supplied by a MilliQ In eg al Wa e
Pu i ica ion Sys em o Ul apu e Wa e , p oduced by Me ck Millipo e (Bu ling on, MA, USA).
2.1. Mic ocon aine s Fab ica ion
Mic ocon aine s we e p oduced on silicon wa e s using he nega i e epoxy-based pho o esis SU-8 wi h wo
s eps o pho oli hog aphy echnique [12]. B ie ly, SU-8 was dispensed on o Si wa e s and spin coa ed in wo
s eps. To emo e he sol en om he esul ing SU-8 ilm, a so -baked s ep was in ol ed and ollowed by
selec i e exposu e o UV ligh h ough a ch omium mask. The polyme iza ion o exposed walls was induced
by pos -exposu e baking. De ining he sidewalls o mic ocon aine s we e achie ed wi h a second spin coa ing
o SU-8, UV li hog aphy, and baking s eps. A e imme sion in p opylene glycol me hyl e he ace a e (PGMEA),
and insing in isop opyl alcohol, mic ocon aine s con aining wa e s we e cu in o 1.2 × 1.2 cm chips using an
au oma ic dicing saw (DAD 321, DISCO, Tokyo, Japan).
2.2. D ug Loading
The mic ocon aine s we e manually illed wi h amoxicillin using he PDMS-masking echnique [13] (Figu e 1).
B ie ly, esin and cu ing agen o Sylga d® 184 we e mixed (10:1 w/w a io) and slowly pou ed in
mic ocon aine -con aining wells in he chip ay. A e he gaps be ween mic ocon aine s ully co e ed by
PDMS, he chip ay was pu in o he o en and kep 30 min a 70 °C o cu ing. Amoxicillin was illed in o he
mic ocon aine s using a spa ula o p ess on o he powde and excess d ug was swep using a so b ush. The
PDMS mask was gen ly peeled o by pulling h ough he edges and de ached om he Si wa e bea ing he
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d ug-loaded mic ocon aine s. The amoun o he d ug was calcula ed by weighing chips be o e and a e
loading.
Figu e 1 Emp y mic ocon aine s on Si chip (a), peeling o PDMS masking a e d ug loading (b), d ug-
loaded mic ocon aine s (c)
2.3. Sp ay Coa ing
Amoxicillin-loaded mic ocon aine s we e coa ed wi h ei he CS, ChS o hei combina ion by using an ul asonic
sp ay coa e (Exac aCoa , Sono Tek, USA) wi h an Accumis nozzle. Fo sp ay coa ing, 0.5% (w/ )
concen a ion o chond oi in sul a e in Milli Q wa e and 0.5% (w/ ) chi osan solu ion in 0.1M ace ic acid we e
p epa ed sepa a ely. The solu ions we e sp ayed on o he mic ocon aine s wi h a 1.3 W gene a o powe and
coa ed in a o al o 100 loops.
2.4. Mo phological Cha ac e iza ion
The mic ocon aine s we e cha ac e ized be o e and a e loading and coa ing p ocesses using a TM3030Plus
able op scanning elec on mic oscope (SEM, Hi achi High Technologies Eu ope GmbH, Ge many) wi h
moun ing on a me allic holde wi h a il o 30° and analyzed a a ol age o 15 keV.
The dimensions o he emp y mic ocon aine s and he hickness o he coa ing we e measu ed using op ical
and con ac p o ilome y using a 3 mg ip o ce wi h a scan speed o 100 μm/s.
2.5. D ug Release
Amoxicillin elease om he coa ed and uncoa ed mic ocon aine s was measu ed using a μ-Diss P o ile (Pion
Inc. Wobu n, MA, USA) wi h a empe a u e-con olled wa e ba h a 37 °C. Each chip was a ached o a
cylind ical magne ic s i e and placed in o a 10mL o PBS- illed glass ial. The abso bance was measu ed a
270 nm. The elease p o iles we e moni o ed in eal- ime h ough in si u UV p obes wi h a pa h leng h o 5mm
and each channel was calib a ed wi h d ug concen a ions o 0.05 o 0.5 mg/mL. The cumula i e elease
p o iles we e calcula ed a each ime poin and p esen ed as mean ± SD.
3. Resul s
Two di e en ypes o p o ilome y we e used o acqui e opog aphical p ope ies o emp y mic ocon aine s
and s ep heigh measu emen s o coa ing. As can be seen om he h ee-dimensional p o iles o he emp y
mic ocon aine s in Figu e 2, hey ha e an inne diame e o 232 ± 1 µm and an inne heigh o 272 ± 3 μm
(mean ± SD, n = 3).
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Figu e 2 2D and 3D p o iles o emp y mic ocon aine s
The mic ocon aine s we e subjec ed o sp ay coa ing o CS o ChS solu ions sepa a ely and in combina ion
wi h he pa ame e s desc ibed in Table 1. Fo he laye -by-laye coa ing, he i s laye was ei he CS o ChS,
and he nex laye consis ed o 50 loops o he coun e pa polysaccha ide ha esul ed in a o al o 100 loops
coa ing. The hicknesses o he di e en coa ings we e measu ed using a s ylus p o ilome e . The highes lid
hickness was ob ained wi h CS due o ha ing a highe densi y, whe eas ChS coa ing was esul ed in a lowe
alue.
Table 1 The pa ame e s o he sp ay coa ing and he hicknesses o he di e en coa ings
Pa ame e s
Coa ings
CS
ChS
CS/ChS
Pa h speed [mm s−1]
20
20
20
In usion a e [mL min−1]
0.05
0.04
0.05/0.04
Gene a o powe [W]
1.3
1.3
1.3
Shaping ai p essu e [kPa]
0.012
0.01
0.012/0.01
Hea pla e empe a u e [°C]
50
35
50/35
No. o loops
100
100
50/50
Thickness (µm)
6.37 ± 1.33
4.49 ± 1.15
5.50 ± 0.74
Figu e 3 SEM images o emp y (a), PDMS-masked d ug loaded (c), a e peeling o he masking (d), CS (e),
ChS ( ) and CS/ChS coa ed mic ocon aine s
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Figu e 3 shows SEM images o uncoa ed, PDMS-masked d ug loading and sp ay coa ed mic ocon aine s. As
can be seen, he d ug loadings we e e icien , he coa ings we e homogenous and success ully sealed he
d ug-loaded mic ocon aine s.
In i o elease p o ile o uncoa ed and polysaccha ides-coa ed amoxicillin loaded mic ocon aine s is shown in
Figu e 4. A cumula i e elease o 97.3 ± 10.3% o uncoa ed, 89.1 ± 3.1% o CS coa ed and 91.5 ± 9.2% o
ChS coa ed mic ocon aine s we e ob ained in 15h. Wi hin he i s hou , an ini ial bu s elease o o e 60% o
amoxicillin occu ed o bo h uncoa ed and ChS con aining mic ocon aine s. Howe e , CS ones showed slowe
elease as can be clea ly seen in he inse o Figu e 4. Ne e heless, CS mic ocon aine s con inued hei as
elease p o ile due o he hyd a ion and swelling, eached and passed uncoa ed and ChS coa ed ones wi hin
2h. Mic ocon aine s wi h CS-ChS and ChS-CS as lids, ollowed signi ican ly slowe and sus ained elease
pa e ns and eached 74.7± 4.8% and 73.8 ± 2.9% a e 14h. This beha io can be a ibu ed o he ac ha
PEC o ma ion be ween CS and ChS would cause he lids o be less suscep ible o swelling and es ain he
elease o amoxicillin [14].
Figu e 4 Cumula i e elease p o ile o amoxicillin om uncoa ed and coa ed mic ocon aine s (mc)
4. CONCLUSION
In his s udy, he in luence o he combina ion o polysaccha ides as polyme ic lids o mic ocon aine s was
e alua ed o an o al d ug deli e y sys em. Following he success ul d ug loading h ough he PDMS masking
echnique, he mic ocon aine s we e coa ed wi h ei he CS, ChS o hei combina ion using sp ay coa ing. SEM
images and hickness measu emen s wi h p o ilome e showed homogenous coa ings o mic ocon aine s. The
esul s o in i o elease s udies con i m ha he mic ocon aine s wi h CS-ChS lids showed a slowe and
p olonged elease ( ega dless o he laye o de ) compa ed o he indi idual coun e pa s, which p o es he
PEC o ma ion be ween he wo polysaccha ides. I is expec ed ha using CS-ChS as elease con olling
polyme lids could be a p omising he apeu ic al e na i e o enhance he o al deli e y o an ibio ics by
inc easing he esidence ime.
ACKNOWLEDGEMENTS
This esea ch unded by In e nal G an Agency o Tomas Ba a Uni e si y in Zlín, Czech Republic
(IGA/CPS/2021/001).

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