Bioadhesi e eu ec ogels suppo ing d ug nanoc ys als o long-ac ing
deli e y o mucosal issues
Ma ía Bea ice Bianchi
a
,
b
, Chunyang Zhang
a
, Elise Ca lin
a
, Giuseppina Sand i
b
,
Ma celo Calde
on
c
,
d
, Eneko La a~
ne a
a
, Ryan F. Donnelly
a
, Ma ías L. Picchio
c
,
*
,
Alejand o J. Pa edes
a
,
**
a
School o Pha macy, Queen's Uni e si y Bel as , Medical Biology Cen e, 97 Lisbu n Road, Bel as , BT9 7BL, UK
b
Depa men o D ug Sciences, Uni e si y o Pa ia, Viale Ta amelli 12, 27100 Pa ia, I aly
c
POLYMAT, Applied Chemis y Depa men , Facul y o Chemis y, Uni e si y o he Basque Coun y, UPV/EHU, Paseo Manuel de La dizabal 3, 20018 Donos ia-San
Sebas i
an, Spain
d
IKERBASQUE, Basque Founda ion o Science, 48009 Bilbao, Spain
ARTICLE INFO
Keywo ds:
Nanoc ys als
Eu ec ogels
Bioadhesion
D ug deli e y
3D p in ing
Cu cumin
ABSTRACT
Eu ec ogels (Egels) a e an eme ging class o so ionic ma e ials ou pe o ming adi ional empe a u e-in ole an
hyd ogels and cos ly ionogels. Due o hei excellen elas ici y, non- ola ile na u e, and adhesion p ope ies, Egels
a e a ac ing a g ea deal o in e es in he biomedical space. He ein, we epo he fi s example o adhesi e Egels
loading d ug nanoc ys als (Egel-NCs) o con olled deli e y o mucosal issues. These so ma e ials we e p e-
pa ed using gela in, glyce ine, a deep eu ec ic sol en (DES) based on choline hyd ochlo ide and glyce ol, and
nanoc ys allised cu cumin, a model d ug wi h po en an imic obial and an i-inflamma o y ac i i ies. We fi s
explo ed he impac o he biopolyme concen a ion on he iscoelas ic and mechanical p ope ies o he ne -
wo ks. Thanks o he dynamic in e ac ions be ween gela in and he DES, he Egel showed excellen s e chabili y
and elas ici y (up o 160%), e e sible gel-sol phase ansi ion a mild empe a u e (50 C), 3D-p in ing
abili y, and good adhesion o mucin p o ein (s ickiness 40 kPa). In i o elease p ofiles demons a ed he
abili y o he NCs-based Egel o deli e cu cumin o up o ou weeks and deposi significan ly highe d ug
amoun s in excised po cine mucosa compa ed o he con ol coho . All in all, his s udy opens new p ospec s in
designing so adhesi e ma e ials o long-ac ing d ug deli e y and pa es he way o explo e no el eu ec ic
sys ems wi h mul iple he apeu ic applica ions.
1. In oduc ion
Deep eu ec ic sol en s (DES) a e an a ac i e class o low-cos elec-
oly es ea u ed by an abno mal dep ession in hei eu ec ic poin em-
pe a u e compa ed o ha o an ideal liquid mix u e [1,2]. The
immobiliza ion o hese in iguing mix u es in polyme ma ixes o e s
new oppo uni ies o gene a ing unc ional gel ma e ials wi h p omising
applica ions in heal hca e [3,4]. Due o he unique ionic conduc i i y o
he DES componen , ecen s udies ha e mainly ocused on explo ing
eu ec ogels as wea able senso s o bioelec odes, aiming o eplace
expensi e and non-biocompa ible ionic liquid gels [5–7]. Howe e ,
eu ec ogels also benefi om se e al ea u es ha can be exploi ed in
con olled d ug deli e y sys ems, such as good biocompa ibili y, adhe-
si eness, s i ness ma ching biological issues, and non- ola ile na u e
[8]. Fu he mo e, DES ha e p o ed o d ama ically inc ease he solubi-
li y, s abili y, and pe meabili y o d ugs, becoming e sa ile deli e y
sys ems o nasal, ansde mal, and o al adminis a ion wi h imp o ed
bioa ailabili y [9–11].
D ug nanoc ys als (NCs) a e sub-mic ome ic pa icles (<1
μ
m)
composed o 100% d ug in a c ys alline s a e, ea u ed by ha ing
inc eased sa u a ion solubili y, dissolu ion a e, and bio-adhesi eness,
allowing o boos he biopha maceu ical pe o mance o hyd ophobic
* Co esponding au ho . POLYMAT Ins i u e - Uni e si y o he Basque Coun y UPV/EHU, Responsi e Polyme The apeu ic G oup, Facul y o Chemis y, Paseo
Manuel de La dizabal 3, 20018, Donos ia-San Sebas i
an, Spain. Tel.: +34 943 01 53 25.
** Co esponding au ho . Lec u e in Pha maceu ical Sciences, School o Pha macy, Queen's Uni e si y Bel as , Medical Biology Cen e, 97 Lisbu n Road, Bel as ,
BT9 7BL, Uni ed Kingdom. Tel: +44(0)2890971061.
E-mail add esses: ma ias.p[email p o ec ed] (M.L. Picchio), [email p o ec ed] (A.J. Pa edes).
Con en s lis s a ailable a ScienceDi ec
Ma e ials Today Bio
jou nal homepage: www.jou nals.else ie .com/ma e ials- oday-bio
h ps://doi.o g/10.1016/j.m bio.2022.100471
Recei ed 13 Sep embe 2022; Recei ed in e ised o m 17 Oc obe 2022; Accep ed 19 Oc obe 2022
A ailable online 25 Oc obe 2022
2590-0064/©2022 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
Ma e ials Today Bio 17 (2022) 100471
d ugs [12–14]. The o mula ion o NCs has become one o he mos
p e e ed echniques o o e coming poo d ug solubili y, bo h in in-
dus ial and academic labo a o ies, allowing o he de elopmen and
app o al o se e al NCs-based medicines sui able o mul iple adminis-
a ion ou es [12,15]. The e o e, combining DES and NCs could esul in
ad an ageous d ug deli e y pla o ms wi h ou s anding ea u es, a oid-
ing he apid clea ance o NCs om he applica ion si e. Consequen ly,
hese echnologies hold subs an ial po en ial o long-ac ing d ug de-
li e y, which is especially appealing in mucosal issues, whe e mul iple
challenges ela ed o mucosae physiology ha e o be o e come [16].
These include a limi ed a ea, sec e ion o fluids, enzyma ic deg ada ion,
poo issue pene a ion, and pala abili y in he case o o al p oduc s [17].
Howe e , sca ce s udies on eu ec ogels as d ug deli e y sys ems ha e
been conduc ed so a , and o he bes o ou knowledge, NCs o mula-
ions ha e no been conside ed in he o mula ion design [18].
Poly(2-hyd oxye hyl me hac yla e) eu ec ogels hos ing choline chlo ide
(ChCl)/asco bic acid (2:1) o ChCl/ uc ose (2:1) DES ha e been ecen ly
de eloped o o al deli e y o suni inib mala e o indome hacin,
espec i ely [19,20]. Fu he mo e, some hyd ogel sys ems inco po a ing
DES as d ug solubilizes o bioac i e liquids ha e been epo ed [21,22].
F ei e e al. p epa ed algina e hyd ogels by adding glyce ol (Gly)/a gi-
nine (4: 1) DES/wa e mix u e as a solubili y enhance o ibup o en [23].
In ano he ecen s udy, amino acids/ci ic acid (3:1) DES we e in e-
g a ed in o Ca bome ®940 hyd ogels o skin deli e y o “Sanwujiao-
wan”ex ac , a Chinese he b medicine [24]. In his con ex , he e is s ill a
p o ound lack o s udies add essing he design o unc ional eu ec ogels
o pha maceu ical and deli e y echnologies.
He ein, we p opose he syne gis ic combina ion o d ug NCs and a
eu ec ic mix u e suppo ed in o a gela in ma ix o ob ain s e chable and
adhesi e eu ec ogels o long-ac ing mucosal deli e y (Fig. 1). Cu cumin
(CUR) was chosen as a model hyd ophobic d ug o p epa e NCs and
glyce ine, ChCl/Gly (1:3), as he eu ec ic sys em. This DES has al eady
demons a ed a powe ul solubili y enhancemen o CUR, p o iding a
g ea op ion o maximize i s he apeu ic ou come [25]. Fi s ly, we
ocused on he e ec o he gela in concen a ion on he eu ec ogels'
iscoelas ic and mechanical p ope ies. The mos p omising o mula ion
was loaded wi h CUR-NCs, and he esul ing eu ec ogels nanocomposi es
we e analyzed in e ms o hei physicochemical p ope ies, 3D-p in ing
abili y, and capabili y o d ug deli e y in an ex i o po cine mucosa
model.
2. Ma e ials and me hods
2.1. Chemicals
Cu cumin (CUR), syn he ic, pu i y >97.0% was pu chased om
Tokyo Chemical Indus ies (London, UK). Gela in om bo ine skin gel
s eng h ~225 g bloom, ype B, choline chlo ide 99% (ChCl), and
glyce ol 99% (Gly) we e ob ained om Sigma Ald ich Co (Gillingham,
UK). Poloxame 188 (P188) was ob ained om BASF Chemical Company
(Ludwigsha en, Ge many). Zi conia beads pa ially s abilized wi h Y ia
( ype YTZP) wi h a diame e o 0.1–0.2 mm we e ob ained om Chemco
(Guang u, China). Ul a-pu e wa e was used in all he expe imen s (Elga
PURELAB DV 25, Veolia Wa e Sys ems, Dublin, I eland). All o he e-
agen s we e o analy ical g ade and pu chased om s anda d comme cial
supplie s.
2.2. P epa a ion o eu ec ogels
The DES was p epa ed by dissol ing ChCl and Gly (1:3 M a io) a 95
C unde s i ing. Once he DES was o med, di e en amoun s o gela in
we e added o he sys em. D ug- ee blank eu ec ogels con aining 10, 15,
20, and 25% w/ o gela in we e p epa ed, wi h hei o mula ion codes
being Egel-10, Egel-15, Egel-20, and Egel-25, espec i ely. A e he
gela in was ully dissol ed, he esul an solu ion was pou ed in o sili-
cone moulds and s o ed o e nigh a 4 C o allow he gel o ma ion. Fo
p epa ing he d ug-loaded eu ec ogels (Egel-NCs), 50 mg o he solid NCs
we e added pe g am o he mol en gel (final d ug concen a ion: 5w
%) and immedia ely blended wi hou hea ing by dual asymme ic
cen i uga ion in a SpeedMixe ™(DAC 150 FVZ, High Wycombe, En-
gland) o 20 s a 3500 pm. The esul an blend was pou ed in o silicone
moulds and allowed o cool a 4 C o e nigh . The ob ained gels we e
s o ed in he idge and p o ec ed om ligh un il used o
cha ac e iza ion.
2.3. Fou ie ans o m in a ed spec oscopy (FTIR)
In a ed spec oscopic pa e ns o CUR, CUR-NCs, and Egel-NCs we e
ob ained using an Accu ac FTIR (FT/IR-4100 Se ies, Jasco, Essex, UK)
equipped wi h Diamond MIRacle™A.T.R. The egion s udied co e ed
he ange o 4000–600 cm
1
wi h he esolu ion se a 4.0 cm
1
. Each
spec um was ob ained using an a e age o 64 epea scans.
2.4. Rheological measu emen s
Ampli ude, equency, and empe a u e sweep expe imen s we e
pe o med in a s ess-con olled An on Paa Physica MCR101 heome e ,
employing 8 and 25 mm pa allel-pla e geome ies. Ampli ude sweep
expe imen s we e ca ied ou om 0.1 o 100% s ain a a cons an e-
quency o 1 Hz a 25 C. F equency expe imen s we e pe o med om 0.1
o 100 ad/s a 1% s ain a 25 C. Tempe a u e sweep expe imen s we e
ca ied ou om 0 o 80 Ca 4C/min and a fixed equency o 1 Hz and
1% s ain (in he linea ange o iscoelas ici y o he ma e ials).
2.5. Tensile es
Fo he ensile es s, gel specimens wi h bone shapes o 25 mm in
leng h and c oss-sec ion 3.5 mm 1 mm we e cu . Tes s we e ca ied ou
using a TA HD plus Tex u e Analyze equipmen (Tex u e Technologies)
a 23 C, 50% ela i e humidi y, and an elonga ion a e o 25 mm/min.
A leas fi e specimens o each sample we e es ed.
2.6. Manu ac u e o CUR nanoc ys als
CUR NCs we e p epa ed by a p e iously epo ed sligh ly adap ed lab-
scale media milling echnique [26]. B iefly, 100 mg o CUR we e placed
in a 10 mL glass ial oge he wi h 5 mL o 0.5, 1, 1.5, o 2% w/ P188
solu ions, 4.5 mL o zi conia beads (Chemco beads, Suzhou, China) and 2
magne ic ba s (25 8 mm), which we e e ically aligned. The sys em
was agi a ed using an RCT BASIC magne ic s i e (IKA, S au en, Ge -
many) a 1200 pm. A e g inding o 24 h, he CUR nanosuspensions
(NSs) we e sepa a ed om he milling media using a 200-mesh sie e. The
esul an NSs we e d ied o 26 h in a eeze-d ye o collec he NCs,
which we e gen ly g ound o a fine powde using mo a and a pes le.
Fig. 1. Schema ic illus a ion o he p epa a ion o gela in-based eu ec ogels
in eg a ing CUR-NCs.
M.B. Bianchi e al. Ma e ials Today Bio 17 (2022) 100471
2
2.7. Pa icle size de e mina ion
The pa icle size o he p is ine d ug was de e mined by lase
di ac ion using a Mas e size ®3000 equipped wi h a Hyd o®cell
(Mal e n Panaly ical L d, Wo ces e shi e, England). Fo his pu pose, a 2
mg/mL suspension o he d ug was p epa ed using P188 2% w/ as he
dispe san . The sample was u he dilu ed un il he sca e ed ligh in-
ensi y was be ween 5 and 15% and analyzed six imes. The esul s we e
exp essed in e ms o he De B oucke e mean diame e ([D4,3]) and
olume densi y dis ibu ion. The pa icle size, polydispe si y index (PDI),
and ze a po en ial o he NCs we e de e mined using a NanoB ook
Omni®analyze (B ookha en, New Yo k, USA). To his end, 5
μ
L o he
NS we e dispe sed manually in 4 mL o wa e , ans e ed o disposable
plas ic cells, and analyzed. The equilib a ion ime was se a 3 min, and
de e mina ions we e made a 25 C. Resul s we e exp essed as mean
alues s anda d de ia ion (mean SD, n¼3).
2.8. P epa a ion o d ug/P188 physical mix u e
A d ug plus P188 physical mix u e (PM) was p epa ed and used as a
con ol h oughou he wo k. The PM con aining 100 mg o CUR and 75
mg o P188 was p epa ed using he SpeedMixe ™ o 5 min a 3500 pm.
This composi ion was equi alen o he d y basis o he nanosuspension
p epa ed wi h 1.5% w/ P188. The esul an powde was used alone o
loaded in o he eu ec ogels (Egel-PM).
2.9. Mic oscopical cha ac e iza ion
The mo phologies o CUR, PM and CUR NCs we e e alua ed using a
Table op TM 3030 scanning elec on mic oscope (Hi achi, Tokyo,
Japan).
2.10. Nanosuspensions s abili y
The physical s abili y o he NSs p epa ed wi h di e en P188 con-
cen a ions was assessed a oom empe a u e and 4 C. Fo his pu pose,
he NSs we e kep in he me ically sealed, ligh -p o ec ed con aine s
placed on he bench op o in he idge, and samples we e aken a imes
0, 7, and 14 days. A e a quick manual edispe sion, 5
μ
L NS aliquo s
we e aken, and he pa icle size, PDI, and ze a po en ial we e measu ed
using he echnique desc ibed in he p e ious sec ion. Resul s we e
exp essed as mean alues s anda d de ia ion (mean SD, n¼3).
2.11. The mal analysis
Di e en ial scanning calo ime y (DSC) and he mog a ime ic
analysis (TGA) we e pe o med on CUR, CUR NCs, and Egel-NCs. DSC
uns we e ca ied ou in Q100 ins umen (TA Ins umen s, New Cas le,
DE, USA) in a empe a u e ange o 25–300 C, wi h a hea ing amp o 10
C/min and a ni ogen flow o 10 mL/min. TGA expe imen s we e ca ied
ou be ween 25 and 500 C wi h a hea ing a e o 10 C/min and a ni-
ogen flow o 10 mL/min, using a Q500 ins umen (TA Ins umen s,
New Cas le, DE, USA).
2.12. Powde X- ay di ac ion
X- ay di ac ion was used o cha ac e ize he pu e d ug, he NCs and
he NCs-loaded eu ec ogels in e ms o c ys alline s a e. Fo his pu pose,
a Miniflex®X- ay di ac ome e was used (Rigaku Co po a ion, Ken ,
England), equipped wi h Cu K
α
adia ion. The scans we e pe o med in a
ange o angles be ween 5and 60in 2θin s eps o 0.04, fixing he
coun ing ime a 0.5 s pe s ep.
2.13. P obe ack es
Adhesion measu emen s we e pe o med using a TA HD plus Tex u e
Analyse equipmen (Tex u e Technologies). Samples o 1.5 mm in
hickness we e fixed on a glass suppo . In he p obe- ack es s, a ci cula
Del in®p obe (10 mm Ø) comes in o con ac wi h he sample a a gi en
eloci y o 1 mm/s. A 500 g comp essi e o ce is applied o 1 s, and he
p obe is emo ed om he gel a a con olled eloci y. Fo he eu ec ogels
adhesion o mucin, a 10 mm Ømucin disc was p epa ed by comp essing
he powde p o ein and a aching i o he p obe ip using an e hyl-
cyanoac yla e adhesi e.
The debonding o ce and displacemen we e eco ded and hen
con e ed o nominal s ess and s ain by no malizing he o ce by he
p obe a ea and he displacemen by he ini ial hickness o he gel,
espec i ely.
2.14. Eu ec ogel e-cas ing and NCs e-dispe sion
The ecycling abili y o he eu ec ogels nanocomposi es was e alu-
a ed by epea ed cycles o hea ing abo e 50 C/cooling a RT o induce
hei gel-sol phase ansi ion. The gels we e e-shaped in di e en o ms
by moulding he ma e ial a he liquid s a e. A e each emoulding cycle,
he edispe sion o he CUR-NCs was assessed by dynamic ligh sca e ing
(DLS).
2.15. 3D p in ing o eu ec ogels
Egel-20 and Egel-20-NCs we e p in ed in o a mesh pa e n using a 3D
bio-p in e (Biosca olde 3.2, GeSiM, Radebe g, Ge many) equipped
wi h a hea ed ca idge and a 0.25 mm nozzle. Bo h ma e ials we e
placed in o he ca idge and hea ed a 90 C o 30 min be o e p in ing.
Then, he empe a u e was allowed o equilib a e a 41 C, and meshes
we e p in ed using a p essu e o 300 kPa. The p in ing speed was 5 mm/
s, and he laye heigh was 0.25 mm. The esul an meshes we e obse ed
in a digi al mic oscope (VHX, Keyence, L d, Mil on Keynes, UK) and a
scanning elec on mic oscope (Table op TM 3030, Hi achi, Tokyo,
Japan). Fu he mo e, DSC expe imen s and edispe sion o CUR-NCs
om he meshes we e pe o med o assess he e ec o he 3D p in ing
on he sys em.
2.16. In i o d ug elease
The in i o elease p ofiles o coa se CUR, PM, CUR NCs and Egel-20-
NCs we e assessed using a dialysis memb ane model acco ding o a
p e iously desc ibed me hodology [27,28]. The elease media consis ed
o 100 mL o 2% w/ Tween®80 in bu e sali a pH 6.4 con aining 0.5%
w/ o asco bic acid o p e en d ug deg ada ion [29]. Fo his expe i-
men , 2.6 mg o CUR powde and CUR NCs (con aining 1.97 mg o CUR)
we e placed in dialysis memb ane bags o 12,000–14,000 kDa molecula
weigh cu -o (MWCO) (Spec a-Po , Spec um Medical Indus ies, Los
Angeles, CA, U.S.A.) and sealed a bo h ends wi h clips. Samples we e
placed in he me ically sealed Du an®bo les, which we e placed in a ISF
7100 o bi al incuba o a 37 C wi h an agi a ion speed o 100 pm 400
μ
L-samples we e wi hd awn a chosen imepoin s (5 h, 24 h, 4 days, 1
week, 2 weeks, 3 weeks, 4 weeks), eplaced wi h esh bu e , and
quan ified by high-pe o mance liquid ch oma og aphy (HPLC). This
expe imen was pe o med in iplica e and he esul s exp essed as
means SD. The d ug elease pe cen ages we e calcula ed using he
ollowing equa ion:
%o d ug eleased ¼CUR quan i ied in he elease media
Ini ial amoun o CUR 100 (1)
2.17. D ug quan ifica ion by HPLC
CUR was quan ified using e e se-phase HPLC on an Agilen ins u-
men equipped wi h UV de ec ion (Agilen Technologies 1220 Infini y
UK L d, S ockpo , UK). The column used was a ZORBAX Eclipse®XDB-
C18 column (50 4.6 mm in e nal diame e ; 1.8
μ
m pa icle size) wi h a
M.B. Bianchi e al. Ma e ials Today Bio 17 (2022) 100471
3
fixed empe a u e o 25 C. The mobile phase comp ised 80% ace oni ile
and 20% phospho ic acid 0.1%. The maxima abso p ion (λ
max
) was fixed
a 425 nm, he injec ion olume was 20
μ
L, and he flow a e was 0.5 mL/
min. The linea i y o he me hod was explo ed in he concen a ion ange
o 0.125–50
μ
g/mL (R
2
¼1), wi h a limi o de ec ion o (LoD) o 0.40
μ
g/
mL and a limi o quan ifica ion (LoQ) o 1.21
μ
g/mL.
2.18. Ex i o mucosal pene a ion
As de ailed below, he ex i o mucosal deposi ion s udies o Egel-20-
NCs and Egel-20-PM we e pe o med in excised buccal neona al and
adul po cine mucosa.
2.18.1. D ug deposi ion in excised adul po cine mucosa
The excised mucosa was ob ained om a local bu che y shop, cleaned
wi h pH 7.4 mucus bu e , and gen ly cu o he size o he F anz cells
dono compa men wi h a s e ile scalpel. Then, 6 mm eu ec ogels disks
we e placed on he mucosa, and he F anz cells' dono sec ion was
a ached o each mucosa using cyanoac yla e glue. Subsequen ly, he
sys em was moun ed on he F anz cells and he ecep o compa men
was filled wi h 12 mL o 2% Tween®80 in PBS wi h 0.5% asco bic acid.
A e wa ds, 100
μ
L o bu e sali a was added in o he dono compa -
men , which was closed using Pa afilm®M o a oid wa e e apo a ion.
The sys em's empe a u e was p ese ed a 37 1C using a wa e
ci cula o (Julabo Co io C, Cole Palme , Ve non Hills, Illinois, USA).
A e 6 h, he sys em was disassembled, he excess o mula ion was
gen ly emo ed, and he mucosa was insed wi h 1.5 mL pH 7.4 mucus
bu e . Subsequen ly, an 8-mm biopsy punch was used o ex ac a sec ion
o he mucosa om whe e he d ug was ex ac ed. An illus a ion o he
expe imen al se up o he mucosal deposi ion in excised neona al mu-
cosa is p o ided in Fig. S1A. This expe imen was ca ied ou in iplica e,
and he esul s we e exp essed as means SD.
2.18.2. D ug deposi ion in excised buccal neona al po cine mucosa
The mucosa was cleaned wi h pH 7.4 mucus bu e and gen ly cu
wi h a s e ile scalpel. Then, as de ailed in p e ious publica ions, each
mucosa sec ion was placed in a weighing boa con aining issue pape
soaked wi h mucus bu e [30]. Resin 3D p in ed ings wi h an inne
diame e o 6 mm and 5 mm heigh we e a ached o he mucosa wi h
cyanoac yla e glue using manual o ce o 30 s. Inside he ing, 6 mm
disks o Egel-20-NCs o Egel-PM we e added, ollowed by 10
μ
L o pH 6.4
sali a bu e . The sys em was sealed wi h Pa afilm M® o a oid wa e
e apo a ion and placed in an o en a 37 1C. A e 24 h, he ings and
eu ec ogels we e emo ed, he mucosa was insed wi h 1 mL pH 7.4
mucus bu e , and he excess o mula ion was wiped wi h a clean issue
pape we ed wi h mucus bu e . Subsequen ly, a 6 mm diame e biopsy
punch was used o ob ain a ci cula sec ion o mucosa om whe e he
d ug was ex ac ed. The manu ac u ing me hod o he ing inse is
p o ided in supplemen a y me hod 1, and an illus a ion o he expe i-
men al se up o he mucosal deposi ion in excised neona al mucosa is
p o ided in Fig. S1B. This expe imen was ca ied ou in iplica e, and
he esul s we e exp essed as means SD.
2.18.3. D ug ex ac ion om mucosal issues
Each ci cula sec ion o mucosa was placed in a 2-mL Eppendo ube
wi h wo s ainless s eel beads (0.5 cm diame e , Qiagen, Hilden, Ge -
many) and 500
μ
L o pu ified wa e . Then he ubes we e placed in a
TissueLyse ®LT (Qiagen, Hilden, Ge many) and p ocessed o 10 min a
50 Hz o hyd a e he mucosa [27,31]. A e wa ds, 1 mL o ace oni ile
was added o each ube, and he p ocess was epea ed o ano he 10 min
a 50 Hz o ex ac he d ug. The homogenized issue was cen i uged a
14,462 g o 10 min (Sigma mic o ube cen i uge SciQuip L d, Sh op-
shi e, UK), and he CUR con en in he supe na an was analyzed by
HPLC using he me hod de ailed in Sec ion 2.18.
2.18.4. Fluo escen mic oscopy
Specimens o he mucosal issues ob ained om he d ug deposi ion
expe imen in excised adul po cine mucosa we e imaged in a mul i-
pho on mic oscope (MPM) (Leica TCS SP8- mul i-pho on exci ed fluo-
escence up igh mic oscope, Leica Mic o-sys ems L d., Mil on Keynes,
UK) o obse e he dis ibu ion o NR in he scle al issue.
2.19. S a is ical analysis
G aphPad P ism©so wa e ( e sion 8.0, G aphPad So wa e Inc, San
Diego, Cali o nia, USA) was used o build plo s and pe o m s a is ical
analyses. An unpai ed - es was applied when compa ing wo coho s,
whe eas one-way ANOVA was applied o compa e mo e han wo co-
ho s. The esul s we e exp essed as means SD, and in all cases, a p-
alue <0.05 deno ed significance.
3. Resul s and discussion
3.1. P epa a ion and cha ac e iza ion o gela in-based eu ec ogels
The ansi ion o gela in chains om andom coil o iple helix a e
hea ing/cooling s eps is a well-known phenomenon inducing gela ion.
Recen s udies ha e shown ha gela in can unde go his ansi ion in
some polyol-based DES, esul ing in eu ec ogels wi h excellen s e ch-
abili y [32,33]. Thus, we selec ed he eu ec ic mix u e glyce ine, ChCl:
Gly (1:3), o d i e he p o ein gela ion. Gela in and Gly ha e shown
excellen compa ibili y, mainly p omo ed by pola in e ac ions be ween
–NH
2
and –COOH g oups om he p o ein and –OH g oups om he
polyol [5]. Indeed, FTIR analysis o Fig. 2A e ealed a significan blue
shi in he C
–
–
O s e ching ( om 1631 o 1650 cm
1
) and N–H bending
om 1525 o 1550 cm
1
) bands o gela in a e eu ec ogel o ma ion.
Fu he mo e, a band b oadening in he egion 3000-3700 cm
1
co e-
sponding o N–H and O–H s e ching is also e idenced. All hese changes
in he ib a ional modes indica e s ong in e molecula in e ac ions,
including hyd ogen bonding, be ween he eu ec ic mix u e and he
p o ein.
Ob iously, he p o ein concen a ion can boos he o ma ion o hese
non-co alen bonds and iple helixes, endowing he biopolyme gels
wi h supe io ea u es. The e o e, we a ied he gela in concen a ion in
he eu ec ogels om 10 o 25% w/ and explo ed he impac on he
heological and mechanical beha iou . As seen in Fig. 2B, ampli ude
sweeps show ha he ma e ials' elas ic modulus (G0) inc eased om 10 o
29 kPa o Egel-10 and Egel-25, espec i ely. Besides, all he eu ec ogels
showed a la ge linea iscoelas ic ange o o e 100%. In he same line,
equency sweeps o Fig. 2C indica e ha he eu ec ogel we e s u dy
ma e ials in he ange o 0.1–100 ad/s, ea u ed by a ma ked inc ease in
G0a high equencies. The s i ening o he eu ec ogel ne wo k can be
a ibu ed o ee gela in chains wi h long elaxa ion imes, ac ing as
physical c osslinke s o he gel ma ix.
Due o he dynamic in e ac ions be ween gela in and Gly, he eu ec-
ogels benefi om a he mo e e sible gel o sol phase ansi ion, which
endows hese ma e ials wi h emoulding and 3D-p in ing abili y. As an
example, he e olu ion o G0, iscous modulus (G00), and an δ s em-
pe a u e o Egel-20 is shown in Fig. 2D. The gel-sol ansi ion empe -
a u e (T
gel-sol
) was defined as he empe a u e a which G' ¼G''. An
inc easing end o he T
gel-sol
wi h he gela in concen a ion was
obse ed, which a ied om 45 o 51 C o Egel-10 and Egel-25,
espec i ely (see Fig. S2 o he supplemen a y In o ma ion). Wi h a
T
gel-sol
o 49 C, Egel-20 was ully s able un il 40 C, he onse o G0,
un eiling he sui abili y o his ma e ial o be used a he body
empe a u e.
Nex , we in es iga ed he mechanical p ope ies o he eu ec ogels by
ensile es , as shown in Fig. 3A. Fig. 3B p esen s he s ess s s ain cu es
ob ained o he eu ec ogels se ies, whe e an elas ic beha iou was
M.B. Bianchi e al. Ma e ials Today Bio 17 (2022) 100471
4
obse ed un il he ailu e o he ma e ials. In addi ion, an e iden
s eng hening e ec o he gela in concen a ion can be obse ed, as he
ensile s eng h and he elonga ion a b eak s eadily inc eased om 11
kPa o 95% o 75 kPa and 167% o Egel-10 and Egel-20, espec i ely
Fig. 2. A- FTIR spec a o glyce ine (ChCl/Gly 1:3), gela in, and Egel-20 eu ec ogel. Ampli ude (B) and equency sweeps (C) o he as-p epa ed eu ec ogels. D-
E olu ion o he dynamic moduli and an δ s empe a u e o Egel-20.
Fig. 3. A- Pho os o ensile es : eu ec ogel a es (le ) and du ing s e ching ( igh ). B- S ess s s ain cu es o eu ec ogels wi h di e en gela in concen a ions.
Elonga ion a b eak s ensile s eng h (C) and Young's modulus s oughness (D) o he as-p epa ed eu ec ogels. Resul s a e exp essed as means SD o ensile
s eng h, elonga ion a b eak, Young's modulus, and oughness (n¼5).
M.B. Bianchi e al. Ma e ials Today Bio 17 (2022) 100471
5
(Fig. 3C). Young's modulus also ollowed he same end anging om 10
o 45 kPa (Fig. 3D), which is in he ange o he s i ness o biological
issues [34].
Due o he almos linea dependence be ween he p o ein concen-
a ion and he ensile s eng h and elonga ion a b eak, Egel-25 esul ed
in he oughes gel wi h a alue o 70 kJ/m
3
(Fig. 3D). Howe e , he
high iscosi y o his o mula ion a he liquid s a e (abo e T
gel-sol
) could
hinde he in eg a ion o d ug NCs, u ning he solu ion unhandy.
The e o e, wi h a compa able oughness alue o 55 kJ/m
3
, Egel-20
was selec ed as he mos p omising ma e ial o suppo he CUR NCs.
The ollowing sec ions will add ess he physicochemical p ope ies o
his d ug nanocomposi e eu ec ogel (Egel-20-NCs).
3.2. Manu ac u e o CUR-NCs
CUR NCs we e success ully ob ained using a simple lab-scale media
milling echnique. The pa icle size dis ibu ion o he coa se d ug ob-
ained by lase di ac ion is p esen ed in Fig. 4A, whe e a mean diame e
D[3,4] o 37.4
μ
m and a bimodal olume dis ibu ion can be obse ed.
This esul was confi med by SEM analysis o he coa se d ug, as shown in
Fig. 4B, whe e d ug pa icles show he e ogeneous sizes and cuboidal
shapes. The milling p ocess was e ficien independen ly o he s abilize
concen a ion, since all he o mula ions p esen ed pa icle sizes below
150 nm (0.5%w/ P188: 128.9 11.9 nm, 1%w/ P188: 87.9 3.0 nm,
1.5%w/ P188: 94.8 3.1 nm, 2%w/ P188: 94.2 6.5 nm). The PDI
alues we e in all cases below 0.260, wi h no subs an ial di e ences
ega ding P188 concen a ion. Also, i is wo h men ioning ha he
milling p ocess did no deg ade he d ug's chemical composi ion, as
demons a ed by FTIR analyses (Fig. S3 o he supplemen a y da a).
Mo eo e , he ze a po en ials we e always nega i e, anging om
12.8 0.9 mV o 15.7 2.2 mV. These esul s indica ed ha he
concen a ion o he s abilize did no play a c i ical ole in he final
p ope ies o he o mula ions a e 24 h o milling. Fig. 4C shows he
pa icle size dis ibu ion o NCs con aining 1.5% w/ o P188, whe e a
monomodal dis ibu ion is obse ed wi h a Z-a e age diame e o 164.4
nm (PDI: 0.201). SEM images o his o mula ion e idenced uni o m
pa icles wi h sizes in good ag eemen wi h DLS esul s (Fig. 4D). The
manu ac u e o NCs using labo a o y media milling echniques has been
p e iously desc ibed, allowing o he p oduc ion o finely dis ibu ed
pa icles in a scalable manne [35]. In his case, he pa icle size esul s
ag eed wi h hose obse ed in p e ious epo s using simila expe i-
men al se ups [36]. Du ing he milling p ocess, he e is an exponen ial
inc ease in he su ace a ea, which gi es NCs some o hei dis inc i e
ad an ages, such as inc eased dissolu ion a e, sa u a ion solubili y and
mucoadhesion [37]. Howe e , NCs mus emain s able o main ain all
hese ad an ages; hence, we s udied he physical s abili y o he di e en
NCs o mula ions.
3.3. S abili y o CUR-NCs
Fig. 5A and Cshows he pa icle size o CUR NCs p oduced using
inc easing concen a ions o P188 a oom empe a u e and 4 C,
espec i ely. Bo h expe imen s showed simila ends, wi h a significan
inc ease in pa icle size a a concen a ion o 0.5% w/ o P188, indi-
ca ing he insu ficien capaci y o he sys em o keep he NCs s abilized
o e he e alua ed pe iod. On he con a y, inc easing concen a ions o
he s abilize led o imp o ed s abili y, wi h negligible changes in he
size, PDI and ze a po en ial o e he e alua ed pe iod a concen a ions o
1.5 and 2% w/ . Mo eo e , he ze a po en ial alues emained nega i e
o he du a ion o he s udy, in all cases wi h alues g ea e han 10
mV, as shown in Fig. 5B and D. Os wald ipening and pa icle agg ega ion
can comp omise he s abili y o he NCs and can be p e en ed by e ficien
s abiliza ion o newly o med su aces du ing he milling p ocess [14,
38]. Gi en he esul s obse ed in p e ious sec ions, he o mula ion
con aining 1.5% w/ o P188 was selec ed o u he s udies.
3.4. Physicochemical beha iou o he nanocomposi e eu ec ogel
DES and NCs o mula ions ha e su p isingly imp o ed he pe me-
a ion and dissolu ion a e o class IV d ugs. He ein, we hypo hesized ha
hese wo echnologies combined could ac syne gis ically, ende ing
inno a i e pha maceu ical pla o ms wi h boos ed deli e y ea u es.
Besides, hei applica ion p ospec is undoub edly b oadened by immo-
bilizing he liquid and nanopa icula e o mula ions in o an elas ic and
adhesi e ma ix.
The s udy o he physicochemical cha ac e is ics o he d ug and he
o mula ion a di e en s ages o he p ocess is help ul o iden i y po-
en ial chemical ins abili y, in e ac ions wi h excipien s, o changes in
Fig. 4. P oduc ion o CUR-NCs by media milling. A- Pa icle size dis ibu ion o coa se CUR ob ained by lase di ac ion; B- SEM images o coa se CUR. C- In ensi y
pa icle size dis ibu ion o CUR-NCs ob ained by DLS. D- SEM images o CUR-NCs.
M.B. Bianchi e al. Ma e ials Today Bio 17 (2022) 100471
6
he c ys alline s a e. Fig. 6A shows he DSC he mog ams o CUR, CUR
NCs and Egel-20-NCs. CUR is clea ly c ys alline as pe he sha p endo-
he mic peak obse ed a 175 C, which co esponds wi h he mel ing
poin o he d ug. A e milling and eeze-d ying, he NCs p esen ed a
mel ing peak a 52 C, co esponding o P188. Mo eo e , he d ug
emained c ys alline in he NCs, bu he endo he mic mel ing peak
shi ed o a lowe empe a u e and dec eased i s in ensi y. This ac has
been shown p e iously in simila o mula ions con aining P188, whe e a
pa ial he mally-induced amo phiza ion occu ed du ing he hea ing
p ocess in he DSC expe imen , and P188 mel s a he ea ly s ages o he
expe imen [14,39,40]. The NCs-loaded eu ec ogel also p esen ed simila
cha ac e is ics, a CUR mel ing peak a a ound 155 C. This peak appea s
a simila empe a u es han he one ob ained o CUR NC o mula ions
indica ing ha his molecule keeps a ce ain deg ee o c ys allini y.
In e es ingly, he DSC cu e shows an addi ional peak a a ound 275 C,
which could be due o he he mal deg ada ion o he sample. Simila
peaks ha e been epo ed o ChCl deg ada ion in he pas [41]. Fu he
he mal cha ac e iza ion is p esen ed in Fig. 6B, whe e he CUR and CUR
NCs powde s p esen ed simila decomposi ion beha iou , s a ing
a ound 300 C. Egel-20-NCs, p esen ed a g adual mass loss un il 150 C,
p obably associa ed wi h mois u e e apo a ion, ollowed by a s eepe
mass loss associa ed wi h he decomposi ion o he sys em. This
decomposi ion is consis en wi h he DSC peaks ob ained a a ound 275
C. Mo eo e , he mal deg ada ion o gela in has been epo ed o s a
be ween 250 and 300 C. In e es ingly, Gly deg ades a highe empe -
a u es [42]. The e o e, i can be es ablished ha he deg ada ion
obse ed in Fig. 6B can be a ibu ed o ChCl and gela in. The X- ay
di ac ion pa e ns (Fig. 6C) demons a e ha he pu e CUR and he NCs
o m o he d ug we e clea ly c ys alline, wi h peaks obse ed ac oss he
e alua ed ange o 2θdeg ees, i.e., a 12, 13, 18, and 24. Expec edly,
Egel-20-NCs showed he ypical halo o amo phous ma e ials, which is
ela ed o he ela i ely la ge p opo ion o amo phous excipien s
compa ed o CUR. Besides, he FTIR analysis shown in Fig. 6D demon-
s a ed a good in e acial in e ac ion be ween he d ug and o he com-
ponen s o he o mula ion. Cha ac e is ic peaks om he CUR NC
o mula ion can be seen in he Egel-20-NCs FTIR spec um, indica ing he
p esence o he d ug in he o mula ion. Finally, i is impo an o high-
ligh ha no signal shi s o new peaks we e obse ed a e gel o ma ion,
sugges ing ha he d ug was no deg aded du ing he o mula ion
p ocess.
In e es ingly, CUR NCs p o ided a ein o cemen e ec in he
eu ec ogel ma ix, inc easing Young's modulus (38 s 55 kPa) and ensile
s eng h (63 s 74 kPa) bu dec easing i s s e chabili y (158 s 129 kPa)
(Fig. 7A). Fu he mo e, equency sweeps (Fig. 7B) showed ha he
inco po a ion o d ug NCs also imp o ed G0in he ubbe y pla eau egion
(blue colou ed) om 18 o 28 kPa. These esul s sugges ha CUR NCs
could ac as physical c osslinking poin s, boos ing he eu ec ogel ne -
wo k's s abili y. Indeed, he empe a u e sweeps in Fig. 7C e ealed an
inc ease in he T
gel-sol
up o 56 C, expanding he applicabili y ange o
empe a u e o he eu ec ogel nanocomposi e.
Since he g ea numbe o pola g oups in gela in and glyce ol
(-COOH, –NH
2
,–OH, e c.), he eu ec ogel nanocomposi e esul ed in a
highly adhesi e ma e ial, as shown in Video S1 o he Supplemen a y
da a. Bioadhesi eness is a key-sough specifica ion o mucus d ug de-
li e y, and hese eu ec ogels could be a ac i e he apeu ic pla o ms o
his applica ion. Nex , we explo ed he adhesion p ope ies o Egel-20-
NCs o mucin p o ein, finding an excellen adhesi e s eng h o 40
7 kPa (Fig. 7D), ou pe o ming o he ecen ly epo ed mucoadhesi e
hyd ogels [43–45].
Supplemen a y da a ela ed o his a icle can be ound a h ps://
doi.o g/10.1016/j.m bio.2022.100471.
Fig. 5. S abili y o CUR-NCs s abilized wi h P188 0.5, 1, 1.5 and 2% w/ . A- Pa icle size and PDI o CUR-NCs a oom empe a u e, B- Ze a po en ial o CUR-NCs a
oom empe a u e, C- Pa icle size and PDI o CUR-NCs a 4 C, D- Ze a po en ial o CUR-NCs a 4 C. Resul s a e exp essed as means SD o PDI and means þSD o
pa icle size (n¼3).
M.B. Bianchi e al. Ma e ials Today Bio 17 (2022) 100471
7
3.5. Eu ec ogel-NCs emoulding and NCs e-dispe sion
One o he mos ema kable cha ac e is ics o hese gela in-based
eu ec ogels is hei abili y o lique y abo e he T
gel-sol
and e-cas in o a
gel o m mul iple imes. We e alua ed he capaci y o he NCs o
edispe se om emoulded Egel-20-NCs. The cas ing and e-cas ing o he
nanocomposi e eu ec ogel using he “Q-U-B00 moulds led o appa en ly
uni o m sys ems wi h well-defined edges and shapes, as shown in Fig. 8A.
The pa icle size o he NCs edispe sed om he fi s , second and hi d
cas ing (co esponding o he Q, U, and B shapes, espec i ely) is
Fig. 6. Physicochemical cha ac e iza ion o CUR, CUR-NCs, and Egel-20-NCs. A- DSC he mog ams, B- The mog a ime ic aces, C- X- ay di ac ion pa e ns, and D-
FTIR p ofiles.
Fig. 7. S ess s s ain cu es (A) and equency sweeps (B) o Egel-20 and Egel-20-NCs eu ec ogels. C- E olu ion o dynamic moduli and an d o he d ug
nanocomposi e eu ec ogel. D- Adhesion cu e o Egel-20-NCs on mucin p o ein as a subs a e.
M.B. Bianchi e al. Ma e ials Today Bio 17 (2022) 100471
8
p esen ed in Fig. 8B. The da a indica e ha no significan di e ences
we e obse ed in he pa icle size o he CUR be ween he fi s and sec-
ond cas ing, whe eas a sligh ly la ge NCs size was obse ed o he hi d
cas ing. Al hough he di e ence in his las case was significan (p¼
0.842), he mean pa icle emained be ween 187 and 213 nm. A simila
end was obse ed o he PDI o he NCs a e edispe sion (Fig. 8C),
wi h alues be ween 0.208 and 0.307, and no significan di e ences
we e obse ed be ween he analyzed samples (p<0.05 in bo h cases).
This da a indica es ha he NCs would be eadily a ailable o dissolu ion
and abso p ion upon con ac wi h biological fluids, e en a e he mal
manipula ion o he gel du ing he manu ac u ing p ocess.
3.6. 3D p in ing o eu ec ogels
Addi i e manu ac u ing is a powe ul echnique o educing he
ime, ma e ial was e, and ene gy needed o ab ica e medical de ices and
is being ac i ely applied o pe sonalized biomedicine [46,47]. The good
s abili y o CUR-NCs a e se e al emoulding s eps o Egel-20-NCs
opens he ga e o explo ing he po en ial o hese e sa ile ma e ials in
3D-p in ing applica ions. Blank eu ec ogels and Egel-20-NCs we e
pa e ned in o wo-laye ed ci cula meshes wi h ex e nal diame e s o 17
mm. Op ical mic oscopy images o he blank eu ec ogels and Egel-20-NCs
can be obse ed in Fig. 9A and B, espec i ely. In bo h cases, he gels
flowed su ficien ly well h ough he biop in e 's nozzle o o m he
designed pa e n. Op ical mic oscopy images e ealed ha he CUR-NCs
we e well dis ibu ed in he blend since he colou was homogeneous
ac oss he sample. C ucially, he hickness o bo h ma e ials allowed he
addi ion o a second laye on op o a fi s p in ed laye , highligh ing he
po en ial o cons uc ing h ee-dimensional objec s based on eu ec o-
gels. This esul ag ees wi h a ecen epo , whe e a semiconduc ing
eu ec ogel was 3D p in ed as a wea able senso [5].
SEM was used o assess he mo phology o he 3D p in ed meshes
made o blank eu ec ogels and Egel-20-NCs, and he images a e p esen ed
in Fig. 10A and B, espec i ely. Bo h o mula ions p esen ed smoo h
su aces wi h he absence o bubbles o pa icle agg ega es. Mo eo e ,
he CUR NCs we e no de ec able in hese images a he used magnifi-
ca ions. The SEM images also showed ha he meshes we e clea ly
o med, wi h uni o m angles and s ands.
Shea s ess and empe a u e applied du ing he p in ing p ocess
could a ec he p in ed ma e ials' s abili y, which makes assessing hei
physicochemical p ope ies c i ical. Fig. 10C shows he DSC aces o he
Egel-20-NCs be o e and a e p in ing, whe e he cha ac e is ic endo-
he mic peaks we e p esen in he e alua ed ange o empe a u es.
Fig. 10D and Eshows he edispe sion o he NCs om he 3D p in ed gels
in e ms o mean pa icle size and PDI, espec i ely. Al hough significan
di e ences we e obse ed be ween samples in e ms o pa icle size, he
edispe sed NCs we e only 16 nm bigge han hose om he o iginal
samples. Simila beha iou is consis en wi h he pa icle sizes ob ained
a e se e al cycles o mel ing and cas ing (sec ion 3.5). The e o e, i is
no s ange o ob ain a sligh ly la ge pa icle size a e hea ing he
samples p io o 3D p in ing. Mo eo e , no significan di e ences we e
obse ed be ween he o iginal and edispe sed samples in e ms o PDI.
3.7. In i o d ug elease and mucosal deposi ion
The elease p ofile o CUR om he pu e d ug, d ug plus P188
physical mix u e (PM), CUR NCs, and Egel-20-NCs is p esen ed in
Fig. 11A. The pu e d ug and PM p esen ed e y simila elease p ofiles,
eaching a maximum o only 10% in 28 days, which indica es he
dissolu ion a e enhancemen ela ed o he s abilize is negligible.
Impo an ly, he inco po a ion o CUR in he PM did no p oduce any
significan e ec on i s physicochemical p ope ies, as e ealed by he
FTIR and pa icle size analyses p esen ed in Fig. S3, and Fig. S4,
espec i ely. CUR NCs and Egel-20-NCs p esen ed subs an ially highe
d ug elease a es, wi h 55.8 2.9% and 41.5 2.3% a day 14,
espec i ely. By he end o he s udy, he amoun o d ug dissol ed om
he NCs-based o mula ions was no significan ly di e en , achie ing a
maximum o 68.2 1.9% and 60.9 6.5% o NCs and he eu ec ogel
o mula ion, espec i ely. C i ically, he Egel-NCs o mula ion did no
ou pe o m he NCs alone, which can be possibly a ibu ed o he
p esence o he Egel ma ix, h ough which he d ug once dissol ed mus
di use in o de o be eleased. CUR is a highly hyd ophobic d ug, and
low elease a es ha e been obse ed p e iously in simila expe imen s
[48]. Howe e , he o mula ion o NCs leads o an exponen ial inc ease
in he specific su ace and, hus, an inc ease in he dissolu ion a e ac-
co ding o he Noyes-Whi ney equa ion [49]. In e es ingly, hese esul s
p o e he abili y o he NC-based o mula ions o long-ac ing d ug
elease.
The mucosal deposi ion in neona al po cine mucosa is p esen ed in
Fig. 8. Egel-20-NCs cas ing mul iple imes and NCs e-dispe sion. Mul iple
cas ing o Egel-20-NCs in o moulds wi h Q-U-B shapes. B- Mean pa icle size o
CUR-NCs a e edispe sion o Egel-20-NCs cas h ee imes, C- PDI o CUR-NCs
a e edispe sion o Egel-20-NCs cas h ee imes. Da a exp essed as means SD
(n¼3).
Fig. 9. Op ical mic oscopy images o 3D p in ed eu ec ogel meshes. A- Meshes
ob ained wi h blank eu ec ogels, B- Meshes ob ained wi h Egel-20-NCs. Scale
ba in all cases: 100
μ
m.
M.B. Bianchi e al. Ma e ials Today Bio 17 (2022) 100471
9
