biomolecules
A icle
How Can Biomolecules Imp o e Mucoadhesion o
O al Insulin? A Comp ehensi e Insigh using
Ex-Vi o, In Silico, and In Vi o Models
Ma iana Ama al 1,†, Ana So ia Ma ins 1,†, JoséCa a ino 2, Ped o Faísca 2, P adeep Kuma 3,
João F. Pin o 1, Rui Pin o 1,4, Isabel Co eia 5, Lia Ascensão6, Rica do A. A onso 7,8,9,
M. Manuela Gaspa 1, Adília J. Cha mie 10 , Isabel Vi ó ia Figuei edo 11,12 and
Ca a ina Pin o Reis 1,13,*
1Resea ch Ins i u e o Medicines (iMed.ULisboa), Facul y o Pha macy, Uni e sidade de Lisboa,
1649-003 Lisboa, Po ugal; [email p o ec ed] (M.A.); [email p o ec ed] (A.S.M.);
j pin o@ .ulisboa.p (J.F.P.); apin o@ .ulisboa.p (R.P.); mgaspa @ .ulisboa.p (M.M.G.)
2Faculdade de Medicina Ve e iná ia, Uni e sidade Lusó ona de Humanidades e Tecnologias/DNA ech
Labo a ó io Ve e iná io, 1749-024 Lisboa, Po ugal; [email p o ec ed] (J.C.);
[email p o ec ed] (P.F.)
3Depa men o Pha macy and Pha macology, School o The apeu ic Sciences, Facul y o Heal h Sciences,
Uni e si y o he Wi wa e s and, Johannesbu g 2193, Sou h A ica; [email p o ec ed]
4JCS. D . Joaquim Cha es, Labo a ó io de Análises Clínicas, 1495-068 Mi a lo es-Algés, Po ugal
5Cen o de Química Es u u al, Depa amen o de Engenha ia Química, Ins i u o Supe io Técnico,
Uni e sidade de Lisboa, A . Ro isco Pais, 1049-001 Lisboa, Po ugal; [email p o ec ed]
6Cen o de Es udos do Ambien e e do Ma (CESAM), Faculdade de Ciências, Uni e sidade de Lisboa,
Campo G ande, 1749-016 Lisboa, Po ugal; [email p o ec ed]
7
CEDOC, NOVA Medical School/Faculdade de Ci
ê
ncias M
é
dicas (NMS/FCM), Uni e sidade No a de Lisboa,
1150-082 Lisboa, Po ugal; [email p o ec ed]
8Ciências Funcionais e Al os Te apêu icos, NOVA Medical School, Faculdade de Ciências
Médicas (NMS|FCM), Uni e sidade No a de Lisboa, 1169-056 Lisboa, Po ugal
9Depa amen o de Física, Faculdade de Ciências e Tecnologia, Uni e sidade No a de Lisboa,
2829-516 Capa ica, Po ugal
10 DREAMS, Uni e sidade Lusó ona de Humanidades e Tecnologias, Campo G ande 376, 1749-024 Lisboa,
Po ugal; [email p o ec ed]
11 Pha macology & Pha maceu ical Ca e, Facul y o Pha macy, Uni e sidade de Coimb a, 3000-548 Coimb a,
Po ugal; [email p o ec ed]
12 Coimb a Ins i u e o Clinical and Biomedical Resea ch (iCBR), Uni e si y de Coimb a,
3000-370 Coimb a, Po ugal
13 IBEB, Biophysics & Biomedical Enginee ing, Faculdade de Ciências, Uni e sidade de Lisboa,
1749-016 Lisboa, Po ugal
*Co espondence: ca a ina eis@ .ulisboa.p ; Tel.: +351-217-946-429 (ex . 14244)
†These au ho s con ibu ed equally o his wo k.
Recei ed: 30 Ma ch 2020; Accep ed: 22 Ap il 2020; Published: 27 Ap il 2020


Abs ac :
Cu en ly, insulin can only be adminis e ed h ough he subcu aneous ou e. Due o he
laws associa ed wi h his ou e, i is o in e es o o ally deli e his d ug. Howe e , insulin deli e ed
o ally has se e al ba ie s o o e come as i is deg aded by he s omach’s low pH, enzyma ic con en ,
and poo abso p ion in he gas oin es inal ac . Polyme s wi h ma ine sou ce like chi osan a e
commonly used in nano echnology and d ug deli e y due o hei biocompa ibili y and special
ea u es. This wo k ocuses on he p epa a ion and cha ac e iza ion o mucoadhesi e insulin-loaded
polyme ic nanopa icles. Resul s showed a sui able mean size o o al adminis a ion (<600 nm by
dynamic lase sca e ing), sphe ical shape, encapsula ion e iciency (59.8%), and high eco e y yield
(80.6%). Ci cula dich oism spec oscopy demons a ed ha p o ein e ained i s seconda y s uc u e
a e encapsula ion. Mo eo e , he mucoadhesi e po en ial o he nanopa icles was assessed in
Biomolecules 2020,10, 675; doi:10.3390/biom10050675 www.mdpi.com/jou nal/biomolecules
Biomolecules 2020,10, 675 2 o 20
silico and he esul s, co obo a ed wi h ex- i o expe imen s, showed ha using chi osan s ongly
inc eases mucoadhesion. Besides,
in i o
and
in i o
sa e y assessmen o he inal o mula ion we e
pe o med, showing no oxici y. Las ly, he insulin-loaded nanopa icles we e e ec i e in educing
diabe ic a s’ glycemia. O e all, he coa ing o insulin-loaded nanopa icles wi h chi osan ep esen s
a po en ially sa e and p omising app oach o p o ec insulin and enhance pe o al deli e y.
Keywo ds:
ma ine-de i ed biomolecules; diabe es melli us; insulin; mucoadhesion; nanopa icle;
o al deli e y
1. In oduc ion
Diabe es melli us is a g oup o me abolic diso de s ha a ise om de ec i e ac ion and/o sec e ion
o insulin, esul ing in hype glycemia [
1
]. Type 1 diabe es is cha ac e ized by he li e-long need o
exogenous insulin eplacemen . These pa ien s ha e he need o sel -adminis e long-ac ing insulin
in o de o es ablish basal le els, and sho -ac ing insulin be o e meals [
2
,
3
]. Insulin adminis a ion
is done by subcu aneous injec ion o by cons an subcu aneous in usions [
3
]. Al hough his ou e
is conside ed he only op ion o insulin he apy, and e y e icien and b oadly used, he e a e
d awbacks and disad an ages associa ed wi h i s usage [4,5]. Some o hese include lipoa ophy and
lipohype ophy on he injec ion si e and discom o [
6
]. In addi ion, all issues a e being exposed
o equal quan i ies o insulin [
7
], being ha insulin eaches he muscles and adipocy es p io o he
li e [
8
], wi h only a ound 20% o he adminis e ed insulin eaching his a ge o gan [
9
]. The pe iphe al
hype glycemia migh lead o unwan ed o e s imula ion o he me abolic esponses [
6
]. When insulin
is adminis e ed subcu aneously, i is i s dis ibu ed o he pe iphe al issues [
10
]. Thus, his ou e
o adminis a ion does no mimic he endogenous insulin p oduced by he isle s o Lange hans by
non-diabe ics. When adminis e ed o ally, exogenous insulin is abso bed in he in es ine, eaching
he li e h ough he po al ein and inhibi ing hepa ic glucose ou pu , mimicking he physiological
pa hway by unde going hepa ic i s passage [
10
,
11
]. Al hough o al deli e y o insulin is he mos
com o able and con enien ou e o he pa ien , i also has di icul ies associa ed wi h i s usage due
o i s p o ein na u e [
12
]. Such di icul ies include poo abso p ion by he in es ine epi helium due
o insulin’s hyd ophilici y and la ge dimensions, as well as insulin deg ada ion due o he pH and
enzymes in he s omach and small in es ine [10,13], leading o low bioa ailabili y [14].
To his da e, many e o s ha e been done o y o imp o e o al adminis a ion o insulin, including
he use o nanopa icles (NPs) [
15
,
16
]. NPs may o e come he men ioned di icul ies by p o ec ing
he p o ein d ug om he hos ile condi ions o he gas oin es inal ac (GIT) and imp o ing i s
abso p ion [
14
]. This can be achie ed by adjus ing he su ace cha ge, shape, size, and hyd ophobici y
o he NPs, and o he cha ac e is ics [
17
]. Fu he mo e, he use o NPs allows con ol o e he
d ug elease [
17
,
18
]. P e ious epo s ha e shown ha using syn he ic and/o na u al polyme s o
nanoencapsula e insulin imp o es i s abso p ion in he in es ine [
17
,
19
]. Mo eo e , by choosing he
co ec ma e ials o p epa e he nano o mula ion, ce ain bene icial cha ac e is ics can be achie ed.
The de eloped insulin o mula ion o o al deli e y en ails insulin-loaded poly (D, L-lac ic-co-glycolic
acid) (PLGA) NPs coa ed wi h chi osan and polye hylene glycol (PEG), wi h an ex e nal coa ing
composed o bo ine se um albumin (BSA). PLGA is widely used as a nanoca ie , because i is
biodeg adable and, when in combina ion wi h polye hylene glycol (PEG), allows o longe plasma ic
ci cula ion ime [
20
]. The addi ion o (BSA), as an ou e coa o he NPs, ac s as a p o ec i e laye agains
p o eoly ic enzymes o he GIT, allowing insulin o each sys emic ci cula ion in ac and inc easing
i s bioa ailabili y a i s abso p ion si e [
21
–
23
], i.e., he in es ine. Bu one o he mos impo an
biomolecules in his wo k comes om he sea. In his case, he ocean has been shown o p o ide a ich
place wi h g ea biodi e si y and chemical en i ies wi h p o en bioac i i ies. Chi osan is gene ally
de i ed om he shells o sh imp and o he sea c us aceans and i ac s as a pe meabili y enhance
Biomolecules 2020,10, 675 3 o 20
by opening he igh junc ions o he in es inal epi helium, acili a ing pa acellula and anscellula
anspo [
23
–
25
]. Mo eo e , chi osan p olongs he esidence o ime o chi osan-coa ed o mula ions
in mucosae, h ough i s in e ac ions wi h mucins [26,27].
This wo k ocuses on p epa a ion and cha ac e iza ion o double-coa ed insulin-loaded NPs by
using he ollowing echniques: dynamic ligh sca e ing and elec opho e ic mobili y o size and
su ace cha ge analysis, espec i ely; scanning elec on mic oscopy o assess he NPs’ su ace; HPLC
o de e mina ion o encapsula ion e iciency; ci cula dich oism o e alua e insulin’s ac i i y a e he
encapsula ion and ex- i o and in silico s udies o access he mucoadhesion. Sa e y assessmen was
in i o
p elimina ily assessed using cells and hen by
in i o
using animal models. Finally, he e icacy
o he o mula ion was
in i o
assessed by e alua ing he e ec o glycemia in diabe ic a s ollowing
o al adminis a ion o he double-coa ed insulin-loaded NPs.
2. Ma e ials and Me hods
2.1. Ma e ials
2.1.1. Chemicals
Plu onic
®
F167 (POLX), pepsin (250 IU/mL), BSA (MW 66 kDa) and PEG 4000 we e acqui ed
om Sigma-Ald ich (S . Louis, MO, USA). PURASORB
®
PDLG 5002- PLGA Ra io L/G% 50:50
(MW 45,000–75,000 Da) was pu chased om Pu ac (Go inchem, The Ne he lands). Chi osan om c ab
shells wi h low molecula weigh (Ald ich), 75–85% deace yla ed, was used [
27
]. The insulin used was
Insuman Rapid (Sano i, Pa is, F ance), a as -ac ing insulin, a a concen a ion o 100 IU/mL. Wa e
MiliQ by Millipo e Co po a ion (Bu ling on, MA, USA). All he chemical p oduc s and sol en s used
a e o analy ic pu i y g ade.
2.1.2. Animals
Male Wis a a s 8–10-weeks old, wi h an a e age weigh o 200 g, we e pu chased om Cha les
Ri e (Ba celona, Spain). Males we e chosen o e emale Wis a a s due o he po en ial in luence
o emale ho mones o e insulin sensi i i y [
28
]. The animal housing was kep a he con olled
empe a u e o 22.0
±
1.0
◦
C, humidi y a 50.0
±
15.0% and a cycle o ligh o 12 h. Animals we e kep
unde s anda d hygiene condi ions, ed wi h comme cial chow and gi en acidi ied d inking wa e
ad libi um. The eed was emo ed 12 h p io o he day o ea men .
All animal expe imen s (P o ocol i le: Imp o emen o insulin o al a ailabili y h ough
encapsula ion in polyelec oly e complex nanopa icles, n.
◦
POCI/SAU-FCF//59940) was conduc ed
in acco dance wi h he EU Di ec i e (2010/63/UE), he Po uguese law (DR 113/2013, 2880/2015 and
260/2016) and he Animal Wel a e Commission o he Facul y o Pha macy, Uni e si y o Coimb a,
app o ed by E hics Commi ee o he Facul y o Pha macy, Uni e si y o Coimb a and by he compe en
na ional au ho i y Di ecção-Ge al de Alimen ação e Ve e iná ia (DGAV).
2.2. Me hods
2.2.1. P epa a ion o NPs
The NPs we e p epa ed acco ding o he modi ied-spon aneous emulsi ica ion sol en di usion
me hod [
29
]. An o ganic solu ion con aining PLGA and insulin was p epa ed in a non-aqueous sol en
mix u e. The la e suspension was g adually added o an aqueous solu ion con aining a su ac an
a 0.1%, POLX, dissol ed in wa e , a pH 4.5, a oom empe a u e (25
◦
C) and s i ed a 800 pm
(Heidolph MR3001, Heidolph Ins umen s, Schwabach, Ge many), o 15 min [
30
]. All pa ame e s
we e conside ed based on p e ious s udies epo ed [
29
,
30
]. Nex , he NPs we e coa ed wi h a chi osan
aqueous solu ion (0.03% w/ , p e iously solubilized wi h glacial ace ic acid a 1%) en iched wi h PEG
(0.150%, w/ ), by mixing he p e ious solu ion wi h an aqueous solu ion con aining hese compounds.
This was done a oom empe a u e, and he NPs we e cons an ly s i ed a a speed o 180 pm,
Biomolecules 2020,10, 675 4 o 20
o 30 min. Then, he coa ed NPs we e e-coa ed wi h a BSA aqueous solu ion, a a concen a ion o
1 g/mL, by s i ing he chi osan-NPs solu ions wi h his aqueous solu ion a a speed o 100 pm, o
30 min. A e he inal coa ing, he o mula ion was cen i uged a 10.000
×
g, o 15 min (Beckman
Ins umen s cen i uge, Inc., B ea, CA, USA), in o de o emo e all he eagen s ha did no eac .
2.2.2. NPs Cha ac e iza ion
Mean Size, Polydispe si y Index (PI), and Ze a Po en ial Analysis
The uncoa ed NPs, chi osan-coa ed NPs and double-coa ed NPs we e cha ac e ized ega ding
hei mean pa icle size, polydispe si y index (PdI) and su ace cha ge as ze a po en ial. Pa icle size
and PdI we e measu ed in dilu ed samples wi h wa e MiliQ (1:10, / ) using Dynamic Ligh Sca e ing
(Ze asize Nano S, Mal e n Ins umen s, Mal e n, Wo ces e shi e, UK), and pe o med in iplica es.
Ze a po en ial was analyzed using an elec opho e ic mobili y assay using he same equipmen , using
NPs dilu ed in wa e MiliQ (same dilu ion).
Su ace and Mo phological Analysis
The mo phology o single-coa ed and double-coa ed NPs was obse ed by Scanning Elec on
Mic oscopy (SEM). Aliquo s (10
µ
L) o pa icle suspensions we e sca e ed o e ound glass co e slips
coa ed wi h poly L-lysine, ha we e p e iously a ached wi h a double ace ape o he mic oscope
s ubs. The samples, a e dying in a desicca o , we e coa ed wi h a hin laye o gold and obse ed on
a JEOL 5200 LV scanning elec on mic oscope (JEOL L d., Tokyo, Japan) a 20 kV. The images we e
digi ally eco ded.
2.2.3. De e mina ion o Encapsula ion E iciency (EE)
The pe cen age o insulin encapsula ed was de e mined indi ec ly by quan i ying he amoun o
insulin p esen in he supe na an a e cen i uga ion o samples (10.000
×
g, 15 min; using a Beckman
Ins umen s cen i uge, Inc., B ea, CA, USA). This quan i ica ion was done by HPLC (Hi achi Sys em
LaC om Eli e, Column o en, Diode A ay De ec o U - is and Pump, Tokyo, Japan), using a Column
Wa e s Symme y C18, 5
µ
m 4.6
×
150 mm, wi h an isoc a ic low o 0.7 mL/min. The mobile phase was
composed by ace oni ile:TFA wa e (60:40) ( / ). The measu emen s we e pe o med in iplica es and
he calib a ion was done wi h a s anda dized solu ion o insulin, a 220 nm wa eleng h. The linea i y
ange was es ablished in he 1.09–70
µ
g/mL ange and he de ec ion limi was 0.359
µ
g/mL and he
quan i ica ion limi was 1.087
µ
g/mL. The e en ion ime was equal o 3.1 min. Encapsula ion e iciency
(EE, %) was hen de e mined by using Equa ion (1):
EE(%)=(Ini ially added insulin −insulin p esen in supe na an )
Ini ially added insulin ×100 (1)
2.2.4. De e mina ion o Reco e y Yield (RY)
NPs we e eco e ed a e being cen i uged and lyophilized a
−
49
◦
C o a leas 48 h (F eezone
2.5 L, F eeze-d ye Labconco, Kansas Ci y, MO, USA). Nex , NPs we e s o ed a 4
◦
C, acco ding o
p e ious wo ks [29]. The eco e y yield (RY, %) was de e mined using Equa ion (2):
RY (%)= inal mass o nanopa icles
mass o componen s used in o mula ion ×100 (2)
Biomolecules 2020,10, 675 5 o 20
2.2.5. Insulin Ac i i y
Ci cula Dich oism (CD)
The seconda y s uc u e o insulin was analyzed by CD spec oscopy. Ci cula dich oism (CD)
spec a we e eco ded on a JASCO J-720 spec opola ime e (JASCO, Hi oshima, Japan) wi h a
180–700 nm pho omul iplie (EXEL-308). CD spec a we e eco ded in he a UV ange om 260 o
200 nm wi h qua z Sup asil
®
CD cu e es (0.1 cm). The measu emen s we e done a ~23
◦
C in a oom
wi h con olled empe a u e. Each CD spec um is he esul o six accumula ions eco ded in deg ees.
The ollowing acquisi ion pa ame e s we e used: da a pi ch, 0.5 nm; bandwid h, 2.0 nm; esponse, 2 s
and scan speed, 50 nm/min. Samples o CD analysis we e ob ained a e dis up ing he chi osan-coa ed
insulin-loaded NPs in PBS (USP 30), pH 7.4 and ul asounds. The insulin concen a ion o e e y sample
was no malized o 1 mg/mL and compa ed wi h equal concen a ions o non-encapsula ed insulin.
The CD signal alues ob ained a 208 nm we e used o es ima e he
α
-helical (%) con en o
he p o ein [
30
,
31
]. CD measu emen s we e exp essed as he mean esidue ellip ici y (MRE in deg
cm2dmol−1), calcula ed om Equa ion (3):
MRE =CD (mdeg)
Cp ×N×l(3)
whe e N is he numbe o amino acid esidues (51 o insulin), l is he leng h o he op ical pa h (0.1 cm)
and C
p
is he concen a ion o he p o ein. The
α
-helical con en (%) is calcula ed om he MRE alues
a 208 nm, using Equa ion (4):
α−helix (%)=−(MRE208nm −4000)
(33000 −4000)×100 (4)
2.2.6. In Vi o Release Assay
A speci ic amoun (10 mg) o double-coa ed insulin-loaded NPs we e placed in 50 mL HCl
(pH 1.2), simula ing gas oin es inal condi ions, and always espec ing sink condi ions acco ding
o insulin’s solubili y. The assay was pe o med a 37
◦
C wi h con inuous s i ing (100 pm), in a
magne ic mul ipla e (Heidolph MR3001, Heidolph, Schwabach, Ge many), o 2 h. Aliquo s (1 mL)
we e collec ed a 0.25, 0.5, 1, and 2 h and eplaced using esh medium o ha e a cons an inal olume.
A e his ime pe iod, he NPs we e cen i uged (
×
g, 10 min) and he pelle was ans e ed o PBS
a pH 6.8. Release assay con inued a a speed o 100 pm, a 37
◦
C, o 6 h. Aliquo s (1 mL) we e
collec ed a 0.25, 0.5, 1, 2, 4, and 6 h, when he assay expe imen was s opped. A he de e mined ime
poin s, he aliquo s we e collec ed and cen i uged (1500
×
g, 10 min), and he pelle was esuspended
in he medium solu ion and e u ned o he elease medium. The aliquo s we e analyzed and he
concen a ion o insulin was de e mined by HPLC ollowing he me hod p e iously desc ibed, in
iplica e, and acco ding o Equa ion (5) [29,31,32]:
Released Insulin (%)=Cn V +Vi Pn−1
i=0Ci
To al mass o he pa icles X d ug con en ×100 (5)
whe e Cn was insulin concen a ion a ime n ( ime poin s), V was o al olume o medium, Vi was
olume o sample collec ed a ime i, and Ci was concen a ion o insulin o sample collec ed a ime i
(ini ial ime poin ).
2.2.7. Ex-Vi o Mucoadhesion S udy
A TA-XTPlus Tex u e Analyse (S able Mic o Sys ems, Godalming, UK) equipped wi h a 5 k load
cell was used o mucoadhesion es s [
33
]. A esh Wis a chemically-induced diabe ic a (explained in
sec ion
in i o
e icacy assay) small in es ine was ha es ed and opened longi udinally, cleaned and

Biomolecules 2020,10, 675 6 o 20
cu in o pieces ha i he mo able cylind ical p obe wi h he lumen side acing ou wa ds, a ached
by a double- ace ape o he p obe. The a in es ine was also placed in a s a ic holde aligned wi h
he p obe, also a ached by double- ace ape. Be ween he in es ine’s po ions and in con ac wi h he
s a iona y pa , he suspensions wi h NPs we e placed: non-encapsula ed insulin, uncoa ed NPs and
double-coa ed NPs a e diges ion wi h pepsin, in equal concen a ions o insulin. The double-coa ed
NPs we e incuba ed wi h pepsin, o 2 h a 37
◦
C, in o de o he albumin o be diges ed, acco ding o
Pha macopeia USP. The simula ed gas ic luid was composed o 3.2 g/L o pepsin (wi h ac i i y o
800 o 2500 uni s pe mg o p o ein), sodium chlo ide, and hyd ochlo ic acid. Du ing he expe imen ,
he mo able pa was lowe ed, un il coming in o con ac wi h he mucosa, up o a o ce o 20 g ,
and hen aised a a cons an speed o 0.25 mm/s.
The displacemen and he o ces o comp ession and de achmen we e eco ded. A cu e o o ce
(g ) e sus ime (s) was ob ained o each expe imen and he peak o ce o displacemen (F
max
, g ) and
a ea o he peak (AUC, g .s) we e acqui ed om his da a. The expe imen s we e pe o med i e imes
o each sample.
2.2.8. In Silico Mucoadhesion Analysis
Fo mucoadhesion analysis o chi osan-coa ed NPs e sus uncoa ed PLGA NPs, ene ge ic and
geome ic s abili y o he polyme -mucin molecula complexes we e de e mined using s a ic la ice
a omis ic simula ions (molecula mechanics simula ions; Chemli e30, Hype cube Inc., Gaines ille,
FL, USA). The s uc u es o PLGA and PEG we e gene a ed as na u al bond angles while he ones o
chi osan and glycosyla ed mucin (MUC) we e gene a ed using he saccha ide building and sequence
edi o ools, espec i ely [
34
]. The indi idual molecules (PLGA, PEG, chi osan, and MUC) as well as he
molecula complexes (PLGA-MUC and chi osan/PEG-MUC) we e ene gy minimized and op imized
using MM+Fo ce Field algo i hm. Fo geome ical op imiza ion, a Polak–Ribie e Conjuga e G adien
me hod was employed un il an RMS g adien o 0.001 kcal/mol was achie ed [35].
2.2.9. P elimina y Sa e y Assessmen
In Vi o Assessmen
The sa e y o double-coa ed insulin-loaded NPs was assessed
in i o
by a MTT assay pe o med
in Caco2 cells, a human in es inal cell line commonly used o his ype o assessmen . These cells
we e kep in Dulbecco’s Modi ied Eagle’s medium (DMEM) high-glucose (4.5 g/L), supplemen ed wi h
10% e al bo ine se um and 100 IU/mL o penicillin and 100
µ
g/mL s ep omycin (he ea e comple e
medium). Cells we e main ained a 37
◦
C, wi h a 5% CO
2
a mosphe e, and checked e e y 2 o 3 days,
un il a con luence o 80% was eached. Then, he cells we e seeded in 96-well pla es, a a concen a ion
o 5.0
×
10
4
cells/mL. The cells we e incuba ed wi h double-coa ed insulin-loaded and emp y NPs,
as well as non-encapsula ed insulin. The concen a ions es ed, o bo h ee and nanoencapsula ed
insulin, anged om 0.0625 o 1 IU/mL, and he equi alen was es ed o double-coa ed emp y NPs.
A e 24 h, comple e medium was emo ed, he cells we e washed wi h phospha e bu e ed saline
(PBS) and he MTT solu ion in incomple e medium (0.5 mg/mL) was added. The cells we e incuba ed
o 4 h. A e he incuba ion ime, Dime hyl Sul oxide (DMSO) was added in o de o dissol e he
o mazan c ys als. Abso bance was measu ed a 590 nm using a BioTek ELx800 Abso bance Mic opla e
Reade (BioTek Ins umen s, Inc., Winooski, VT, USA).
In Vi o P elimina y Sa e y Assessmen
The
in i o
p elimina y sa e y assessmen was pe o med in 18 male Wis a a s, weighing
app oxima ely 200 g. The animals we e andomly sepa a ed in o i e g oups: The es g oup (n =5),
o which 50 IU/kg o double-coa ed insulin-loaded NPs was o ally adminis e ed; he ehicle con ol
g oup (n =5), which ecei ed emp y double-coa ed NPs, by o al ga age; he nega i e con ol (n =2),
which ecei ed PBS o ally; he o al insulin con ol (n =3), ha ecei ed 50 IU/kg o comme cial insulin
Biomolecules 2020,10, 675 7 o 20
no inco po a ed in NPs, o ally; and inally, he con ol o insulin’s ac i i y (n =3), o which 4 IU/kg
o comme cial insulin was subcu aneously adminis e ed. A e 6 h, u ine samples we e collec ed
om all animals. The animals we e hen eu hanized, plasma was collec ed, o hema ological and
biochemical analyses, and spleen, s omach, li e , in es ine and kidney we e ha es ed o his ologic
analysis. The o gans we e ixed in 10% o malin and embedded in pa a in. Fi e mic ome e sec ions
o each o gan we e p epa ed o hema oxylin-eosin s aining. The s ained slices we e examined unde
an Olympus BX51 mic oscope (Olympus Co po a ion, Tokyo, Japan) and images we e cap u ed wi h
NanoZoome -SQ Digi al slide scanne (Hamama su Pho onics, Hamama su Ci y, Japan). The u ine
samples collec ed we e es ed o leukocy es, u obilinogen, bili ubin, hema u ia, ni i es, pH, densi y,
p o einu ia, glycosu ia, and ke onic bodies, using U i es 10 V U inalysis Reagen S ips. Plasma
samples we e es ed o quan i y IL-6, ALT ( o de e mine li e oxici y), c ea ine and u ea ( o de e mine
kidney oxici y).
2.2.10. In Vi o E icacy Assay
Diabe es Melli us Induc ion
In o de o chemically induce diabe es melli us in he male Wis a a s, s ep ozo ocin (STZ),
p epa ed in ci a e bu e 0.1 M a pH 4.5 (UPS 30), was adminis e ed in ape i oneally (i.p., 65 mg/kg
o body weigh ). A e his p ocedu e, animals we e exposed o a 5% glucose solu ion du ing he nigh ,
in o de o a oid eac ional hypoglycemia, caused by he STZ. The animals we e classi ied as diabe ic
when glycemia was highe han 300 mg/dL, measu ed on he hi d day pos -adminis a ion [
31
,
36
].
The expe imen al p o ocol was s a ed 10 days a e STZ adminis a ion [29,36].
S udy Design
Fo he
in i o
e iciency assay (n =14), animals we e andomly di ided in o h ee g oups: The es
g oup (n =5), in which 50 IU/kg o NPs o mula ion was o ally adminis e ed; he nega i e con ol
g oup (n =3), o which emp y NPs we e adminis e ed; in he hi d g oup, insulin was adminis e ed
o ally (n =6). The o mula ion e icacy is ansla ed by dec easing glycemia, glycemia was measu ed
a he ollowing ime poin s: 30 min, 1, 2, 4, 6, and 8 h. Glycaemia le els we e de e mined by
measu ing glucose oxidase/pe oxidase, using a glucosome e (OneTouch
®
Ve io
®
IQ, Milpi as, CA,
USA). The animals we e hen sac i iced. The plasma o which he double-coa ed insulin-loaded NPs
we e adminis e ed was collec ed and i s insulinaemia was de e mined by elec ochemiluminescence
immunoassay/(Roche-Cobas®).
2.2.11. S a is ical Analysis
Each alue is p esen ed wi h a mean alue
±
SD. The s a is ical di e ences we e e alua ed
wi h -S uden es and ANOVA. These es s allow o compa e wo o mul iple g oups, espec i ely.
All analyses we e conduc ed in G aphPad P ism Ve sion 5.03 (G aphPad So wa e, San Diego, Cali o nia,
USA) and he di e ences we e deemed signi ica e a a p<0.05.
3. Resul s
3.1. NPs Cha ac e iza ion: Size, Su ace Cha ge, PI, Mo phology, EE, and RY
Mean size and PdI a e shown in Table 1. Insulin-loaded NPs ha e a la ge mean pa icle size
han emp y NPs. I is assumed ha he inc ease in pa icle size was ela ed o he encapsula ion o
insulin. Besides he pa icle size, he e a e o he impo an pa ame e s ha con ibu e o an inc ease
abso p ion o he NPs in he in es inal mucosa, such as NPs cha ge. This p ope y was e alua ed by
measu ing he NPs ze a po en ial in all phases o p oduc ion, also shown in Table 1. A ep esen a i e
scheme o insulin NPs is displayed in Figu e 1. The uncoa ed emp y and uncoa ed insulin-loaded
NPs (Figu e 1A) showed a nega i e su ace cha ge. This alue was cohe en wi h he PLGA cha ge
Biomolecules 2020,10, 675 8 o 20
a he conside ed pH, as p e iously epo ed [
37
]. The NPs cha ge was in e ed o posi i e, in bo h
emp y NPs and insulin-loaded NPs by coa ing he NPs wi h chi osan, a ca ionic polyme , and PEG
(Figu e 1B). The inal o mula ion was ob ained a e he BSA coa ing. The cha ge o he NPs coa ed
wi h BSA, bo h emp y and insulin-loaded double-coa ed NPs (Figu e 1C), emained posi i e, al hough
sligh ly less posi i e. These esul s we e as expec ed and cohe en wi h wha was desc ibed in p e ious
s udies [23,38].
Table 1.
Mean size, PdI and ze a po en ial h oughou he di e en s eps in o mula ion, o bo h emp y
and insulin-loaded NPs. All da a is p esen ed as mean ±SD (n =3).
Sample Mean Size
(nm) PdI
Mean
Ze a Po en ial
(mV)
Emp y NPs
Uncoa ed NPs 240 ±2 0.110 ±0.016 −39 ±6
Chi osan-coa ed NPs 328 ±2 0.231 ±0.015 +48 ±8
Double-coa ed NPs 233 ±2 0.184 ±0.016 +34 ±8
Insulin-loaded NPs
Uncoa ed NPs 936 ±4 0.449 ±0.023 −49 ±5
Chi osan-coa ed NPs 819 ±7 0.527 ±0.028 +41 ±13
Double-coa ed NPs 560 ±9 0.546 ±0.030 +31 ±3
Figu e 1.
Double-coa ed insulin-loaded nanopa icles (NPs) h oughou he o mula ion and coa ing
p ocesses: (
A
) uncoa ed insulin-loaded poly (D, L-lac ic-co-glycolic acid) (PLGA) NPs; (
B
) chi osan-coa ed
insulin-loaded PLGA NPs; and (C) double-coa ed insulin-loaded PLGA NPs.
Figu e 2shows he images ob ained by scanning elec on mic oscopy o bo h double-coa ed
emp y and insulin-loaded NPs, whe e NPs seem o ha e a well-de ined sphe ical shape.
Figu e 2.
SEM mic og aphs o double-coa ed NPs. (
A
) Emp y NPs. (
B
) Insulin-loaded NPs. No e in
bo h cases he well-de ined NPs sphe ical shape. Scale ba s =5µm.
The EE was 59.8 ±2.6%, o he insulin used in he ini ial o mula ion. The RY was 80.6 ±1.1%.
Biomolecules 2020,10, 675 9 o 20
3.2. Insulin Seconda y S uc u e
Ci cula dich oism (CD) was used o e alua e he seconda y s uc u e o nanoencapsula ed
insulin, a e being eleased, which was shown o emain in ac , as illus a ed in Figu e 3, whe e i is
obse ed he cha ac e is ic spec a o a p o ein wi h an alpha helix s uc u e. Figu e 3p esen s CD
spec a showing wo peaks, which a e cha ac e is ic o insulin’s CD spec um, a 211 nm and 222 nm.
An es ima e o he
α
-helical con en was done using he MRE a 208 nm. This yielded he ollowing
alues 31%, 28%, and 27% o non-encapsula ed insulin, uncoa ed NPs and chi osan-coa ed NPs,
espec i ely. Thus, only a small dec ease in he
α
-helical con en was de ec ed along he p epa a ion and
coa ing o NPs, sugges ing no insulin ib illa ion/agg ega ion among he condi ions es ed. Mo eo e ,
hese indings we e also suppo ed by HPLC (obse ing he same e en ion ime o insulin in all
samples) and hen a e in i o o al adminis a ion (Sec ion 3.7).
Figu e 3.
Ci cula dich oism (CD) spec a o non-encapsula ed insulin (
___
), uncoa ed NPs (—) and
chi osan-coa ed NPs ( . . . ) in he Fa UV ange.
3.3. In Vi o Release Assay
This assay was pe o med in o de o be e unde s and insulin’s elease p o ile in he
gas oin es inal ac , and he esul s a e shown in Figu e 4. Tempe a u e was kep a 37
◦
C and s i ing
was always cons an . Fi s ly, he nanoencapsula ed insulin was kep in acidic medium and aliquo s
we e collec ed up o 2 h. In he acidic medium simula ing gas ic condi ions, chi osan is p obably
s a ing o be dissol ed a acidic pH and insulin elease was 25.6
±
3.4% a e 2 h. Fo he neu al
medium (mimicking he in es ine), NPs a e less coa ed and insulin immedia ely s a ed o elease and
i was 100% eleased a e 4 h.
Figu e 4.
Release assay o he double-coa ed insulin-loaded NPs in acidic medium un il 2 h and
conduc ed in neu al medium om 2 o 8 h, mimicking he pH ange o he gas oin es inal ac (GIT).
Biomolecules 2020,10, 675 16 o 20
s abiliza ion was signi ican ly highe o chi osan/PEG-MUC (
∆
E
≈ −
54 kcal/mol) han o PLGA-MUC
(
∆
E
≈ −
7.5 kcal/mol). These esul s con i m he p e e able mucoadhesi e p o ile o chi osan/PEG-coa ed
NPs as compa ed o he uncoa ed PLGA NPs, as p edic ed in he ex- i o esul s.
The de elopmen o sui able and biocompa ible d ug deli e y sys ems is a p e equisi e, especially
o a ch onic disease like diabe es [
17
]. MTT assay was done o assess cy o oxici y and exhibi ed good
esul s as i demons a ed ha double-coa ed insulin-loaded NPs did no al e cell iabili y. Rega ding
emp y NPs, he lowes mean alue o iabili y ound was 85%. This alue o cell iabili y o he
emp y NPs could be a ibu ed o he NPs’ s uc u e by i sel as p e iously demons a ed [
54
], and i is
no no iceable in insulin-loaded NPs possibly due o he insulin’s p esence, since insulin can ac as a
g ow h ac o , p omo ing cell g ow h.
In i o
assays a e ex emely use ul ools, bu hey canno accu a ely p edic he
in i o
beha io
o all o mula ions. The e o e, an
in i o
p elimina y sa e y assessmen s udy was also pe o med.
To assess NPs o mula ion sa e y when o ally adminis e ed, animals’ beha io was closely moni o ed
du ing he es . No dea h occu ed a e he ea men . When compa ing he u ine es s esul s,
biochemical analysis and his ological images o insulin-loaded NPs and nega i e con ol g oup
(PBS g oup), i is displayed ha he e was no ob ious damage o animals. Thus,
in i o
s udies esul s
we e in ag eemen wi h
in i o
MTT assay, i.e., ou insulin-loaded NPs a e sa e o o al adminis a ion.
The p esen ed esea ch en ails o de elop an insulin o mula ion sui able o o al deli e y as
an al e na i e ea men o he comme cially a ailable subcu aneous insulin. O al adminis a ion is
conside ed as he bes ou e o adminis a ion because o i s cos -e ec i eness and well-es ablished
accep abili y. In addi ion, i allows a oiding he use o injec ions. An addi ional ad an age is ela ed o
a mo e physiological ac ion by i s di ec e ec on hepa ic glucose p oduc ion. I he insulin would be
abso bed in he gu , insulin would be ans e ed di ec ly owa d he li e . A he li e , he exogenously
insulin would con ol hepa ic glucose p oduc ion o he same ex en simila ly as his is induced by
endogenously insulin in heal hy subjec s. This mo e physiological deli e y would be associa ed wi h
educed pe iphe al hype insulinemia in con as o SC adminis a ion. In e ms o pha macokine ics,
as ep esen a i e example, in a small s udy made by Ce nea e al. 2004 [
55
], male subjec s unde
euglycemic condi ions, o al insulin sp ay was associa ed wi h a highe Cmax, sho e T
max
, and as e
ime o peak glucose up ake compa ed wi h SC insulin. The sho T
max
and he 120-min du a ion
o e ec o o al insulin sp ay sugges i may be a p omising al e na i e o ul illing meal- ela ed
insulin equi emen s in pe sons wi h diabe es. Ano he encou aging s udy was done wi h a ew
numbe o subjec s wi h 8 mg o o al insulin o mula ion [
56
]. Adminis a ion o an o al o m o
insulin in he as ed s a e demons a ed a signi ican e ec on insulin abso p ion. This subs an ial
e ec was seen by a educ ion o blood glucose (7%–37%), decline in C-pep ide le els (13%–87%),
as well as an ele a ion o insulin le el (20%–120%). I was no iced ha some subjec s de eloped
symp oma ic hypoglycemia. Insulin o mula ions we e well ole a ed. No ad e se o se ious ad e se
e en s ha e been epo ed. The e o e, we aimed o de elop a new o al insulin o ma ion and o
e alua e i he de eloped o mula ion is e ec i e when adminis e ed o ally. The esul s showed ha
he o mula ion was e ec i e in educing glucose le els o diabe ic a s (Figu e 9). A p e ious s udy
compa ing he subcu aneous adminis a ion o 4 IU/kg insulin wi h o al adminis a ion o 50 IU/kg o
nanoencapsula ed insulin coa ed wi h chi osan and albumin in Wis a diabe ic a s has shown ha he
NPs o mula ion achie ed a educ ion in glycemia o 28% be ween he 2nd and 4 h hou , and 48%
be ween he 8 h and 12 h hou [
29
]. When compa ed o o he s udied o mula ions, he o mula ion
epo ed he ein has shown p e e able cha ac e is ics. Many o he p e iously de eloped o mula ions
o o al deli e y o insulin ha e nega i e su ace cha ge [
50
–
54
], no a o ing he NPs’ in e ac ions
wi h mucins, and hus no p omo ing adhe ence o he in es ine’s mucosa [
10
,
22
,
23
]. Besides ha ing a
posi i e su ace cha ge, he double-coa ed insulin-loaded NPs showed o in e ac wi h mucin, he e o e
p o ing i s mucoadhesi e p ope ies, as obse ed bo h by he in silico simula ions o molecula
in e ac ion conduc ed in acuum, and by he ex i o expe imen s. The double-coa ed o mula ion
also showed lowe insulin elease in gas ic-like condi ions a e 2 h (22.6%) han o he o mula ions

Biomolecules 2020,10, 675 17 o 20
in ended o o al deli e y o insulin wi h hal and mo e han hal o he encapsula ed insulin being
eleased in s omach-like condi ions a e wo hou s (~50% [
57
], ~60% [
58
]), sugges ing ha , unlike o he
o mula ions, ou double-coa ed insulin-loaded NPs o mula ion has he po en ial o deli e insulin in
he p ope a ge — he gu . Mo eo e , ou o mula ion was mo e e icien in lowe ing diabe ic- a s han
o he s: while ou double-coa ed insulin loaded NPs induced a 50% glycemia dec ease 8 h pos -o al
adminis a ion, o he s udied o mula ions epo ed smalle and la e hypoglycemic e ec s, o less
han 60% a e 12 h [
59
] o 24 h [
60
] pos -o al adminis a ion. Fu he mo e, when compa ed o o he
chi osan-based insulin-loaded NPs, he de eloped o mula ion had highe encapsula ed e iciency,
was mo e esis an o gas ic-like condi ions o /and achie ed highe dec ease in glycemia, a 4 h, han
wha was seen o o he chi osan-based o mula ions o insulin nanopa icles [61–63].
5. Conclusions
O al insulin eplacemen he apy emains a e y appealing al e na i e o subcu aneous injec ions
o pa ien s wi h diabe es melli us. Howe e , i seems ha he sea ch o an accep able insulin o al
o mula ion is much mo e di icul han ini ially hough . A e decades o ailed a emp s o p oduce
an o al insulin o mula ion, he numbe o published clinical ial epo s so a is limi ed. The hope
clea ly is o see mo e clinical da a. A sui able d ug ca ie is impo an o ensu e si e-speci ic sus ained
d ug deli e y.
As desc ibed he ein, ou double-coa ed insulin-loaded NPs o mula ion showed o be e icien
in educing he glycemia up o 50% in chemically-induced diabe ic a s and i is sa e. Addi ionally,
he encapsula ion was e ec i e and he me hod o encapsula ion did no al e he insulin’s seconda y
s uc u e. So, i is expec ed ha he d ug main ains i s in eg i y when i eaches i s biological a ge .
Fu he mo e, i was p o ed in he in i o assays ha by encapsula ing insulin and coa ing he NPs
wi h wo di e en laye s, insulin was p o ec ed om he hos ile en i onmen o he GIT. Mo eo e ,
he ex- i o s udy showed ha he chi osan coa ing exe s i s mucoadhesi eness, co ela ing wi h he
in silico analysis.
Thus, hese double-coa ed insulin-loaded NPs migh p o ide a mo e e icien and sa e pla o m
o deli e ing insulin by mimicking physiologic p ocesses and di ec ly deli e insulin o he li e
a he han ia he bloods eam. A g ea amoun o wo k s ill emains o be done, bu like many o he
examples in o he he apeu ic a eas, nanomedicine b ings new hope o succeed in ob aining an o al
ea men o insulin a ailable o diabe ic pa ien s.
Au ho Con ibu ions:
Concep ualiza ion, C.P.R.; Me hodology: C.P.R., M.A., A.S.M.; Fo mal Analysis, M.A.,
A.S.M., P.F., P.K., J.F.P., R.P., I.C., L.A., M.M.G., R.A.A., I.V.F., R.P., C.P.R.; In es iga ion, M.A., A.S.M., J.C., P.F.,
P.K., R.P., I.C., L.A., M.M.G., I.V.F., C.P.R.; W i ing – O iginal D a P epa a ion, M.A., A.S.M.; W i ing – Re iew &
Edi ing, P.F., P.K., J.F.P., R.P., I.C., L.A., M.M.G., R.A.A., I.V.F., C.P.R.; Supe ision, C.P.R.; P ojec Adminis a ion,
C.P.R.; Funding Acquisi ion, A.J.C. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
Suppo ed in pa by UID/DTP/04138/2019 om FCT, Po ugal and DREAMS (ULHT). SEM analysis
was unded by FCT/MCTES o he inancial suppo o CESAM (UIDP/50017/2020+UIDB/50017/2020), h ough
na ional unds.
Acknowledgmen s:
The au ho s a e g a e ul o he Ca la V
â
nia (iMedUlisboa) o he collabo a ion in HPLC
analysis and Joana Mo ei a (ECTS-ULHT) o he collabo a ion in conduc ing some expe imen s.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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