biomolecules
A icle
Bioins uc i e Laye -by-Laye -Coa ed Cus omizable 3D P in ed
Pe usable Mic ochannels Embedded in Pho oc osslinkable
Hyd ogels o Vascula Tissue Enginee ing
C is iana F. V. Sousa †, Ca a ina A. Sa ai a †, Tiago R. Co eia , Tamagno Pesquei a, Sónia G. Pa ício,
Ma ia Isabel Rial-He mida , João Bo ges * and João F. Mano *
Ci a ion: Sousa, C.F.V.; Sa ai a, C.A.;
Co eia, T.R.; Pesquei a, T.; Pa ício,
S.G.; Rial-He mida, M.I.; Bo ges, J.;
Mano, J.F. Bioins uc i e
Laye -by-Laye -Coa ed Cus omizable
3D P in ed Pe usable Mic ochannels
Embedded in Pho oc osslinkable
Hyd ogels o Vascula Tissue
Enginee ing. Biomolecules 2021,11,
863. h ps://doi.o g/10.3390/
biom11060863
Academic Edi o : Mikhail
A. Anisimo
Recei ed: 13 May 2021
Accep ed: 7 June 2021
Published: 10 June 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
CICECO–A ei o Ins i u e o Ma e ials, Depa men o Chemis y, Uni e si y o A ei o,
Campus Uni e si á io de San iago, 3810-193 A ei o, Po ugal; [email p o ec ed] (C.F.V.S.);
[email p o ec ed] (C.A.S.); co [email p o ec ed] (T.R.C.); [email p o ec ed] (T.P.);
[email p o ec ed] (S.G.P.); [email p o ec ed] (M.I.R.-H.)
*Co espondence: [email p o ec ed] (J.B.); [email p o ec ed] (J.F.M.); Tel.: +351-234-372-585 (J.B.);
+351-234-370-733 (J.F.M.)
† These au ho s con ibu ed equally.
Abs ac :
The de elopmen o complex and la ge 3D ascula ized issue cons uc s emains he majo
goal o issue enginee ing and egene a i e medicine (TERM). To da e, se e al s a egies ha e been
p oposed o build unc ional and pe usable ascula ne wo ks in 3D issue-enginee ed cons uc s o
ensu e he long- e m cell su i al and he unc ionali y o he assembled issues a e implan a ion.
Howe e , none o hem ha e been en i ely success ul in a aining a ully unc ional ascula ne wo k.
He ein, we epo an al e na i e app oach o bioenginee 3D ascula ized cons uc s by embedding
bioins uc i e 3D mul ilaye ed mic ochannels, de eloped by combining 3D p in ing wi h he laye -
by-laye (LbL) assembly echnology, in pho opolyme izable hyd ogels. Algina e (ALG) was chosen
as he ink o p oduce cus omizable 3D sac i icial mic os uc u es owing o i s biocompa ibili y and
s uc u al simila i y o he ex acellula ma ices o na i e issues. ALG s uc u es we e u he LbL
coa ed wi h bioins uc i e chi osan and a ginine–glycine–aspa ic acid-coupled ALG mul ilaye s,
embedded in shea - hinning pho oc osslinkable xan han gum hyd ogels and exposed o a calcium-
chela ing solu ion o o m pe usable mul ilaye ed mic ochannels, mimicking he biological ba ie s,
such as he basemen memb ane, in which he endo helial cells we e seeded, deno ing an enhanced
cell adhesion. The 3D cons uc s hold g ea p omise o enginee ing a wide a ay o la ge-scale 3D
ascula ized issue cons uc s o modula TERM s a egies.
Keywo ds:
biocompa ible polyme s; 3D p in ing; laye -by-laye assembly; pe usable mul ilaye ed
mic ochannels; na u al-o igin hyd ogels; endo helial cells; p e ascula ized ne wo ks; modula
issue enginee ing
1. In oduc ion
The abili y o bioenginee biomime ic 3D issue-like bio unc ional ascula cons uc s
o accu a ely ec ea e li ing issue-speci ic ascula a chi ec u es and hei physicochemical,
biomechanical and biological unc ions is he majo and long-s anding goal o bo om-up
issue enginee ing and egene a i e medicine (TERM), aiming o eplace, es o e and/o
egene a e damaged issues and o gans [
1
–
6
]. Con en ionally, hyd ogels a e known as he
gold s anda d 3D pla o ms o cell encapsula ion, con olled he apeu ics deli e y and
cons uc ion o 3D issue-like cell-bioma e ial sca olds o bo om-up TERM, owing o hei
nume ous appealing ea u es, including biocompa ibili y, highly hyd a ed 3D en i onmen ,
unable physicochemical p ope ies, mechanical simila i y o na i e issues and ease o
implan a ion ia minimally in asi e p ocedu es [
7
–
10
]. To da e, unc ional issues, such
as ca ilage, bladde and skin, ha e been success ully enginee ed and ansla ed in o
Biomolecules 2021,11, 863. h ps://doi.o g/10.3390/biom11060863 h ps://www.mdpi.com/jou nal/biomolecules
Biomolecules 2021,11, 863 2 o 16
he clinical p ac ice by eso ing o hyd ogel-based cons uc s [
11
–
14
]. Howe e , hei
e ec i eness in sus aining cell iabili y is limi ed o mic osized sys ems. La ge hyd ogels
(abo e ca. 200
µ
m) deno e an inhomogeneous cell dis ibu ion and poo long- e m and
con olled di usion o oxygen, nu ien s, and me abolic was e emo al due o hei inabili y
o build spa ially o ganized pe usable ascula ne wo ks, hus leading o cell apop osis and
he o ma ion o nec o ic co es ha dic a e hei ailu e upon implan a ion and p e en hei
clinical ansla ion [
15
]. As such, enginee ing complex and la ge ascula ized unc ional
issue cons uc s emains elusi e, being essen ial o main ain issue heal h.
O e he las wo decades, signi ican p og ess has been made in he enginee ing o
3D na u al and syn he ic hyd ogel-based sca olds, embedding pe usable mic ochannel
ne wo ks ha a e o be seeded wi h cells, which imp o e hei mass anspo p ope ies o
sus ain long- e m cell iabili y, and enable he o ma ion o complex and la ge 3D ascula -
ized issue cons uc s by eso ing o a wide a ie y o mic o ab ica ion echniques [16].
Among hem, sac i icial molding echniques, including so li hog aphy and eplica
molding [
17
–
23
], and mo e ecen ly 3D (bio)p in ing [
24
–
35
] o combina ions o
he eo
[25,36–38]
, ha e been widely employed o enginee pe usable mic o luidic ne -
wo ks o con olled sizes and geome ies wi hin hyd ogel sca olds o gene a ing unc-
ional ascula ized issue-enginee ed cons uc s. Howe e , he o me is labo ious, cos ly,
ime-consuming and di icul o scale-up. In addi ion, i is mos ly applied o he ab ica ion
o 2D plana pe usable mic ochannels ha do no ec ea e he complex 3D issue a chi-
ec u e, and mos commonly eso s o he use o cy o oxic o ganic sol en s o ha m ul
p ocessing condi ions, including ex eme empe a u e o emo ing he sac i icial em-
pla es, hus no being complian wi h biomolecules. Mo eo e , despi e he emendous
g ow h and exci ing p og ess deno ed by he 3D (bio)p in ing echnology o e he las
ew yea s, i s ill has limi a ions on p in ing esolu ion, choice o (bio)ma e ials and li ing
cells, con olled cell dis ibu ions and ascula iza ion, and hus do no enable enginee ing
3D cons uc s ac oss all scales and ully ec ea ing he complexi y o na i e issues [
39
,
40
].
In con as , he key enabling ea u es endowed by he laye -by-laye (LbL) assembly
echnology u n i in o a p omising al e na i e o enginee bioins uc i e pe usable mi-
c ochannels exhibi ing mul i unc ionali ies. This echnology has p o en o be a simple,
cos -e ec i e, biologically sa e and highly e sa ile bo om-up app oach o con o mally
coa any ype o su ace and p ecisely enginee highly hyd a ed and hie a chical ex acel-
lula ma ix-mime ic bioma e ials, wi h ined- uned s uc u es, p ope ies and unc ions a
he nanoscale, by eso ing o a my iad o biological componen s exhibi ing complemen a y
in e ac ions [
41
–
50
]. Howe e , he lack o a ca ie pla o m ha could encapsula e, p o ec
and endow he de eloped bioma e ial s uc u es wi h supe io biomechanical s abili y o
be adminis e ed in he human body ia minimally in asi e p ocedu es ex ensi ely limi s
he use o he LbL echnology on i s own.
He ein, we p opose a new gene a ion o bioenginee ed, cy ocompa ible and cus omiz-
able 3D pe usable bioins uc i e mul ilaye ed mic ochannels embedded in pho opolyme -
izable na u al-o igin hyd ogels o p omo ing he o ma ion o mic o ascula ne wo ks in
3D enginee ed- issue cons uc s
in i o
, o be po en ially used in modula TERM s a egies.
The cy ocompa ible ee o m mic ochannels we e p epa ed by 3D p in ing biocompa ible
algina e (ALG) sac i icial empla es. ALG biopolyme was chosen as he ink o p oduce
he cus omizable 3D p in ed s uc u es due o i s widely and eadily a ailabili y, p o en
biocompa ibili y, non-cy o oxici y and non-immunogenic p ope ies [
51
]. Mo eo e , he
ionically c osslinked ALG co e empla e can be easily lique ied, unde mild condi ions,
using e hylenediamine e aace ic acid (EDTA), hus u ning i in o a e y appealing sac i i-
cial empla e o being coa ed wi h bioins uc i e mul ilaye ed ilms in a LbL ashion and
enginee hollow mul ilaye ed nanos uc u es o ul illing biomedical pu poses. The 3D
p in ed ALG sac i icial empla e s uc u es we e con o mally LbL su ace unc ionalized
wi h bioins uc i e mul ilaye ed hin ilms endowed wi h cell adhesion mo i s, namely, chi-
osan (CHT)/a ginine–glycine–aspa ic acid (RGD)-g a ed ALG bilaye s. The LbL-coa ed
ALG s uc u es we e u he embedded in a shea - hinning, pseudo-plas ic suppo ing p e-
Biomolecules 2021,11, 863 3 o 16
hyd ogel ma ix encompassing biocompa ible and biodeg adable glycidyl me hac yla ed
xan han gum (XG-GMA) [
52
], o enginee he bioac i e and obus 3D cons uc s a e UV
ligh -induced pho oc osslinking. The exposu e o he 3D cons uc o an EDTA aqueous
solu ion led o he lique ac ion o he ALG pe missi e co e, gene a ing bioins uc i e
hollow mul ilaye ed mic ochannels embedded wi hin he XG-GMA suppo ing hyd ogel
ma ix o he cul u e o human umbilical ein endo helial cells (HUVECs). P e ious wo ks
ha e p oposed he use o he LbL me hod o ecapi ula e biological ba ie s, such as he
basemen memb ane [
53
,
54
]. Howe e , o he bes o ou knowledge, i is he i s ime ha
ubula s uc u es made om mul ilaye s a e embedded in a suppo ing hyd ogel ma ix
as pe usable mic ochannels. We hypo hesize ha his s a egy could assign a a ie y
o p ope ies o he bioenginee ed cons uc , including mass anspo and mechanical
mic oen i onmen con ol be ween he hyd ogel and he mic ochannel, as well as exposu e
o biochemical ac o s (ei he on he su ace o encapsula ed in he mul ilaye s) o he cells.
The
in i o
biocompa ibili y o he de eloped bioac i e 3D cons uc s was s udied, e eal-
ing an enhanced cell iabili y o HUVECs seeded on he bioac i e LbL- unc ionalized
mic ochannels, holding g ea p omise o he gene a ion o bio unc ional ascula ne -
wo ks. The high e sa ili y impa ed by he combina ion o 3D p in ing, LbL assembly
echnology and pho oc osslinkable hyd ogels enables he ab ica ion o cus omizable and
bioins uc i e pe usable mul ilaye ed mic ochannels, po en ially opening new a enues
o con olled he apeu ics deli e y, as well as o enginee ing a wide a ay o la ge-scale
3D ascula ized issue cons uc s o modula TERM s a egies.
2. Ma e ials and Me hods
2.1. Ma e ials
Sodium ALG de i ed om b own algae (Mw = 538 kDa, iscosi y
≈
250 cP) was
pu chased om Sigma-Ald ich (S . Louis, MO, USA) and used as ecei ed. ALG-RGD
(NOVATACH
TM
MVG GRGDSP pep ide-coupled ALG) was pu chased om No aMa ix
(Sand ika, No way) and used as ecei ed. CHT o medium molecula weigh
(Mw = 236.8 kDa, 80% deg ee o deace yla ion, iscosi y
≈
390 cP) was kindly p o ided by
P imex EHF (Siglu jo du , Iceland) and used wi hou u he pu i ica ion. XG (
2–20 MDa
)
was pu chased om Do es Fa m Foods L d. (dis ibu ed by B ice Unipessoal, Po -
ugal). Phospha e bu e ed saline (PBS), sodium hyd oxide (NaOH), calcium chlo ide
(CaCl
2
), EDTA, 2-hyd oxy-4
0
-(2-hyd oxye hoxy)-2-me hylp opiophenone (I gacu e 2959),
hodamine B iso hiocyana e (RITC), dispase II, collagenase ype IV, Medium 199 (M199)
and gela in Type B om bo ine skin we e pu chased om Sigma-Ald ich (S . Louis, MO,
USA). Glacial ace ic acid (CH
3
COOH) and hyd ogen pe oxide (H
2
O
2
) we e pu chased
om JMGS (Odi elas, Lisboa, Po ugal) and Ca lo E ba (Sabadell, Ba celona, Spain), e-
spec i ely. GMA (>95%, s abilized wi h hyd oquinone monome hyl e he (MEHQ)) was
pu chased om TCI Chemicals (New B unswick, NJ, USA). Hepa in was pu chased om
PanReac AppliChem ITW Reagen s (Da ms ad , Ge many). All o he eagen s, namely
ammonium hyd oxide (NH
4
OH), dime hyl sul oxide (DMSO), Glu aMAX
™
-I, e al bo ine
se um (FBS), penicillin, s ep omycin and li e/dead ki we e pu chased om The mo
Fische Scien i ic (Fai Lawn, NJ, USA). All he aqueous solu ions we e p epa ed using
ul apu e wa e om a Milli-Q Plus wa e pu i ica ion sys em ( esis i i y >18.2 M
Ω
cm)
om Me ck Millipo e (Bu ling on, MA, USA).
2.2. P epa a ion o ALG Ink
An ALG ink was p epa ed ollowing he me hod desc ibed by F eeman and Kelly,
wi h sligh changes in he ink iscosi y [
55
]. B ie ly, an ALG ink was p epa ed a 5%
(w/ ) in PBS. Mo eo e , a 60 mM CaCl
2
aqueous solu ion was p epa ed o ionically p e-
c osslink he ALG ink by mixing he wo solu ions a a olume ic a io ( / ) o 25:9 (ALG
solu ion:CaCl2solu ion) o 30 min. Then, he ALG ink was eady o be p in ed.
Biomolecules 2021,11, 863 4 o 16
2.3. 3D P in ing o ALG Ink
3D solid ALG mic os uc u es we e designed wi h compu e -aided design (CAD)
so wa e (SolidWo ks S uden S anda d (SSS) 2020). ALG s uc u es wi h di e en sizes
and geome ies we e 3D p in ed using a 23G needle ip (diame e = 330
µ
m). The size
o he ALG s uc u es anged om 1 o 5 cm, acco ding o he complexi y o he ALG
s uc u es o be p in ed. A e wa ds, he CAD designs we e assembled using 3D p in ing
so wa e (Repe ie -Hos 2.0.5) and, ollowing he axes calib a ion, he p e iously ALG ink-
loaded ca idges we e i ed and placed in o he p in e ’s ca idge holde . The p in ing
p ocess was pe o med a oom empe a u e, wi h an ex usion p essu e o 50 kPa and
a needle speed o 25 mm s
−1
using an Ink edible+ 3D biop in e (Cellink, Go henbu g,
Sweden). Then, he 3D p in ed ALG designs we e imme sed in a 60 mM CaCl
2
aqueous
solu ion o 5 min o ionically pos -c osslink he ALG s uc u es ollowed by insing wi h
ul apu e wa e .
2.4. Syn hesis o RITC-Labeled CHT
RITC-labeled CHT was syn hesized by p epa ing a 1% (w/ ) CHT aqueous solu ion
in 0.1 M ace ic acid, ollowed by he addi ion o anhyd ous me hanol unde s i ing o
3 h. The eac ion p oceeded o 4 h in he da k a oom empe a u e. Then, he RITC-
labeled CHT was p ecipi a ed in 0.2 M NaOH and washed wi h me hanol (70%) un il
he supe na an was luo escence- ee. RITC-CHT was dissol ed in 0.1 M ace ic acid and
dialyzed agains deionized wa e o 7 days using a 6–8 kDa MWCO egene a ed cellulose
memb ane (Spec um Labo a o ies Inc., Rancho Dominguez, CA, USA). The whole eac ion
and dialysis pu i ica ion p ocess we e pe o med p o ec ed om ligh . Finally, RITC-CHT
was eeze-d ied and s o ed a 4 ◦C un il use.
2.5. Ze a (ζ)-Po en ial Measu emen s
Be o e he build-up o he mul ilaye ed hin ilms, he elec opho e ic mobili y o
he eshly p epa ed 0.5 mg mL
−1
CHT, ALG and ALG-RGD aqueous solu ions (pH 5.5)
was s udied by measu ing hei ze a (
ζ
)-po en ials. The
ζ
-po en ials o he indi idual
solu ions we e measu ed using a Ze asize Nano-ZS (Mal e n Ins umen s L d., Roys on,
He o dshi e, UK) a 25
◦
C. The elec opho e ic mobili y (u) was con e ed in o a
ζ
-
po en ial alue ollowing he Smoluchowski equa ion (
ζ
=u
η
/
ε
, whe e
η
and
ε
s and o
he iscosi y and pe mi i i y o he solu ion, espec i ely) [
56
]. The measu emen s we e
pe o med in iplica e and a e aged o each sample.
2.6. Qua z C ys al Mic obalance wi h Dissipa ion Moni o ing (QCM-D)
The g ow h o he bioac i e, elec os a ic-d i en CHT/ALG-RGD mul ilaye ed hin
ilms on o he gold-coa ed 5 MHz AT-cu qua z c ys al senso s (QSX301 Gold, Q-Sense,
Sweden) was moni o ed in si u by QCM-D (QSense P o, Biolin Scien i ic, Go henbu g,
Sweden). Be o e ha , he gold-coa ed qua z c ys al subs a es we e ho oughly cleaned
wi h an oxidizing cleaning solu ion encompassing a 1:1:5 ( / ) mix u e o NH
4
OH (25%),
H
2
O
2
(30%) and ul apu e wa e in an ul asonic ba h a 70
◦
C o 10 min. The Au-
pla ed qua z c ys al senso s we e hen ho oughly insed wi h ul apu e wa e a oom
empe a u e, d ied unde a so s eam o N
2
and submi ed o UV/Ozone (UV/Ozone
P oCleane 220, BioFo ce Nanosciences, Inc., Ames, IA, USA) ea men o 10 min. The
eshly cleaned senso s we e inse ed in he QCM-D appa a us and equilib a ed in an
aqueous solu ion (pH 5.5) un il a s able baseline was achie ed. A e wa ds, he gold-pla ed
qua z senso s we e al e na ely exposed o a 0.5 mg mL
−1
CHT (6 min adso p ion ime)
and RGD-coupled ALG (6 min adso p ion ime) aqueous solu ions a pH 5.5, ende ing
he subs a e su ace posi i ely and nega i ely cha ged, espec i ely. In-be ween he
deposi ion o he opposi ely cha ged biopolyme ic ma e ials, he subs a es we e insed
wi h an aqueous solu ion (pH 5.5) o 4 min o emo e loosely adso bed molecules. The
assembly p ocess was epea ed six imes un il eaching (CHT/ALG-RGD)
6
mul ilaye ed
hin ilms. The inal (CHT/ALG-RGD)
6
mul ilaye ed hin ilms we e d ied unde a so
Biomolecules 2021,11, 863 5 o 16
s eam o N
2
. The expe imen s we e pe o med a a cons an low a e o 50
µ
L min
−1
and a 25
◦
C. The gold-pla ed qua z c ys al subs a es we e exci ed a mul iple o e ones
(1, 3, 5, 7, 9 and 11 co esponding o 5, 15, 25, 35, 45 and 55 MHz, espec i ely) and he
changes in he equency (
∆
) and dissipa ion (
∆
D) we e moni o ed in eal ime. Mo eo e ,
he equency o each o e one was no malized o he undamen al esonan equency
(5 MHz) o he qua z c ys al subs a e (∆ n/n,n e e s o he o e one numbe ).
The hyd odynamic hickness o each o he adso bed laye s was es ima ed using he
Voig -based iscoelas ic model implemen ed in he Q-Sense D ind so wa e (B oad i unc-
ion), assuming a luid iscosi y o 1 mPa s and a luid and laye densi y o 1000 kg m−3.
2.7. Build-Up o 3D P in ed ALG-Templa ed Bioins uc i e Polysaccha ide-Based Mul ilaye ed
Thin Films
The build-up o he bioac i e (CHT/ALG-RGD)
6
mul ilaye ed hin ilms on o he
3D p in ed ALG- empla ed mic os uc u es was pe o med by epea ing he LbL al e -
na e imme sion o he ALG-p in ed sac i icial s uc u es in 1 mg mL
−1
CHT and ALG-
RGD aqueous solu ions (pH 5.5) o 6 min each using a home-made au oma ic dipping
obo (CORPUS
®
, Guima ães, Po ugal). In-be ween he deposi ion o he opposi ely
cha ged biopolyme s, he ALG- empla ed mic os uc u es we e insed wi h an aqueous
solu ion a pH 5.5 o 4 min o emo e he loosely adso bed molecules and a oid he c oss-
con amina ion o he biopolyme ic solu ions. The deposi ion cycles we e epea ed six imes
un il eaching a (CHT/ALG-RGD)
6
mul ilaye ed ilm. By he end o he assembly p ocess,
he bioins uc i e (CHT/ALG-RGD)
6
LbL- unc ionalized, 3D p in ed ALG mic os uc u es
we e kep in wa e a 4
◦
C un il used, o p e en hem om d ying and collapsing. The
p epa a ion o he (RITC-CHT/ALG)
6
mul ilaye s empla ed on 3D p in ed ALG sac i icial
empla es was pe o med in a eminiscen ashion, aiming o p o e he success ul and
con o mal LbL coa ing by luo escence mic oscopy (Axio Image M2 up igh wide ield
luo escen mic oscope, Ca l Zeiss, Jena, Ge many).
2.8. P epa a ion o Bioac i e 3D Cons uc s
Bioac i e 3D cons uc s encompassing bioins uc i e (CHT/ALG-RGD)
6
mul ilay-
e ed ilms unc ionalized 3D p in ed ALG sac i icial mic os uc u es embedded in pho-
oc osslinkable XG-GMA suppo ing hyd ogel ma ices we e p epa ed, p e en ing he
collapsing o he LbL- unc ionalized mic os uc u e a e ALG co e empla e lique ac ion
and he cul u e o HUVECs in he hollow mic ochannels o enginee p e ascula ne wo ks.
2.8.1. Syn hesis o XG-GMA
The chemical modi ica ion o he XG polyme backbone wi h pho opolyme izable
me hac yla e g oups was pe o med as p e iously desc ibed [
57
,
58
]. B ie ly, a 0.5% (w/ )
XG aqueous solu ion was p epa ed in deionized wa e , unde igo ous s i ing, a oom
empe a u e. GMA (5 mL) was added o his solu ion d opwise and he mix u e was
s i ed o 12–18 h a 80
◦
C. Then, he un eac ed molecules we e emo ed by dialysis
agains deionized wa e o 3 days, a oom empe a u e and in he da k, using a 6–8 kDa
MWCO egene a ed cellulose memb ane (Spec um Labo a o ies Inc., Rancho Dominguez,
CA, USA). Finally, he esul ing solu ion was ozen a
−
80
◦
C and eeze-d ied (Tels a
LyoQues Plus Eco, VWR). The lyophilized XG-GMA was s o ed a 4
◦
C, in he da k,
un il use. The chemical cha ac e iza ion o he ob ained XG-GMA compound has been
p e iously epo ed by ou g oup, con i ming he e ec i e unc ionaliza ion o he XG
biopolyme backbone wi h GMA [58].
2.8.2. P epa a ion o Pho ocu able XG-GMA P e-Hyd ogels
The pho ocu able XG-GMA p e-hyd ogel aqueous solu ion was p epa ed by solubiliz-
ing lyophilized XG-GMA in PBS (pH 7.4) con aining 0.1% (w/ ) o I gacu e 2959 o a inal
concen a ion o 0.5% (w/ ).
Biomolecules 2021,11, 863 6 o 16
2.8.3. P epa a ion o Pho oc osslinkable XG-GMA Hyd ogels Embedding Pe usable
Mic ochannels
The ob ained XG-GMA p e-hyd ogel aqueous solu ions we e pou ed in o a silicone
mold and he 3D p in ed ALG empla e sac i icial mic os uc u e, ei he non- unc ionalized
o LbL unc ionalized wi h he bioins uc i e (CHT/ALG-RGD)
6
mul ilaye ed hin ilm,
was embedded on i , lea ing bo h ends o he ALG mic os uc u e ou side he XG-
GMA p e-hyd ogel solu ion a opposing ends. Then, he XG-GMA p e-hyd ogel solu-
ion was c osslinked by exposu e o UV ligh i adia ion (OmniCu e S2000,
320–500 nm
,
100 mW cm−2
, Mississauga, ON, Canada) o 60 s. The pho oc osslinkable XG-GMA hy-
d ogel, embedding ei he he uncoa ed o he bioac i e LbL- unc ionalized 3D p in ed
ALG- empla ed mic os uc u e, was imme sed in a 10 mM EDTA aqueous solu ion (pH 8)
o e nigh o lique y he 3D p in ed ALG co e s uc u e and ob ain a hollow mul ilaye ed
mic ochannel wi hin he XG-GMA hyd ogel.
2.8.4. Rheological Cha ac e iza ion o he Pho oc osslinkable XG-GMA Hyd ogels
The heological cha ac e iza ion o he XG-GMA p e-hyd ogel suppo ing ma ix a
0.5% (w/ ) was pe o med a 25
◦
C on a Kinexus Lab+ heome e (Mal e n Panaly ical,
Mal e n, UK), equipped wi h a UV cu ing chambe i ed wi h a 20 mm-diame e pa allel
pla e geome y and a gap size o 500
µ
m. The shea - hinning p ope ies we e s udied
by measu ing he a ia ion in he iscosi y wi h a con inuously amped shea a e (0.1
o 100 s
−1
). The linea iscoelas ic egion (LVER) was de e mined by measu ing he
s ain ampli ude sweep (0.1 o 1000%) a a equency o 1 Hz. Oscilla o y equency
sweep es ing (0.01 o 10 Hz) was conduc ed a 1% s ain ampli ude. To demons a e he
a ia ion in he XG-GMA elas ic modulus du ing he pho oc osslinking p ocess, a ime
sweep measu emen was ca ied ou a 1 Hz wi h UV ligh exposu e (OmniCu e S2000,
320–500 nm, 100 mW cm−2, Mississauga, ON, Canada).
2.9. In Vi o Cell Cul u e
2.9.1. Cell Isola ion and Cul u e
HUVECs we e e ie ed om umbilical co d ob ained unde a coope a ion ag eemen
es ablished be ween CICECO – A ei o Ins i u e o Ma e ials, Uni e si y o A ei o and
Cen o Hospi ala do Baixo Vouga (A ei o, Po ugal), a e app o al by he Compe en
E hics Commi ee (CEC). The ecei ed human issues we e handled in acco dance wi h
he guidelines app o ed by he CEC and in o med consen was ob ained om all subjec s.
HUVECs we e isola ed ollowing an well-es ablished p o ocol in he g oup [
59
]. B ie ly,
an enzyma ic mix u e con aining dispase II and collagenase ype IV was used o isola e
he HUVECs om he umbilical co d. The co d ein was illed wi h he enzyme cock ail.
Mul iple si e injec ions ou in he co d ma ix using he enzyme cock ail we e also ca ied.
Subsequen ly, he co d was incuba ed a 37
◦
C o 20 min, and hen he HUVECs we e
seeded in M199 g ow h medium ollowed by hei incuba ion in a humidi ied a mosphe e
o 5% CO
2
a 37
◦
C. A e 4–6 h, he medium was eplaced by esh M199 con aining 20%
umbilical co d blood se um, 2 mM L-glu amine, 5 ng mL
−1
ascula endo helial g ow h
ac o , 10 µg mL−1hepa in, 100 U mL−1penicillin, and 100 µg mL−1s ep omycin [60].
P io o HUVECs cul u e, a T75 cell cul u e lask was coa ed wi h a 2% gela in solu ion
and le o incuba e o a leas 30 min, a 37
◦
C, in a humi ied a mosphe e con aining
5% CO
2
. Then, he gela in laye was cleaned wi h s e ile PBS. Cells we e seeded in he
p e-coa ed T75 cell cul u e lask a a densi y o 1
×
10
6
cells/mL and incuba ed a 37
◦
C
in a humi ied a mosphe e con aining 5% CO
2
. The cul u e medium was changed e e y
2–3 days
. Cell p oli e a ion was moni o ed using a ligh mic oscope (Axiocam 105 colo ,
Ca l Zeiss, Jena, Ge many).
Biomolecules 2021,11, 863 7 o 16
2.9.2. Cell Seeding and Viabili y wi hin he Pe usable Mic ochannels Embedded in 3D
P in ed XG-GMA Hyd ogels
Upon eaching he con luence, HUVECs we e ypsinized. New cell suspensions
we e p epa ed a a densi y o 2
×
10
6
cells/mL in M199 cul u e media supplemen ed
wi h and wi hou FBS. Then, cells we e seeded in o ei he he LbL- ee o (CHT/ALG-
RGD)
6
LbL- unc ionalized hollow mic ochannels embedded in he XG-GMA hyd ogels
o 4 h a 37
◦
C in a humi ied 5% CO
2
a mosphe e o allow endo helial cells o adhe e
o he mic ochannels. To e alua e he cell iabili y a e 3 days o cul u e, he XG-GMA
hyd ogels embedding ei he he uncoa ed o (CHT/ALG-RGD)
6
LbL- unc ionalized hollow
mic ochannels we e incuba ed in a calcein-AM/p opidium iodide (PI) solu ion o 30 min.
The o e all 3D cons uc s we e imaged using con ocal lase scanning mic oscopy (CLSM;
LSM 880 Ai y Scan, Zeiss, Jena, Ge many). The acqui ed da a we e p ocessed in Zeiss ZEN
3.0 blue edi ion so wa e. All HUVECs used we e be ween passages 5 and 7 o ensu e he
ep esen a ion o key endo helial cell cha ac e is ics. The quan i ica ion o he cell iabili y
was pe o med using he ImageJ so wa e (Fiji 1.52n).
3. Resul s and Discussion
In his wo k, bioac i e 3D cons uc s encompassing (i) bioins uc i e LbL- unc ionalized
3D p in ed mul ilaye ed mic ochannels embedded in (ii) biocompa ible and biodeg adable
pho oc osslinkable XG-GMA hyd ogels we e de eloped aiming o p e ascula ne wo ks.
B ie ly, biocompa ible ALG co e mic os uc u es exhibi ing di e en sizes and geome-
ies we e success ully 3D p in ed. Such s uc u es we e u he LbL unc ionalized wi h
mul ilaye s encompassing biocompa ible, non-cy o oxic, non-immunogenic and eadily
a ailable ma ine-o igin polysaccha ides, namely, posi i ely cha ged CHT and nega i ely
cha ged ALG modi ied wi h he cell-adhesi e pep ide sequence RGD. The bioins uc i e
LbL-coa ed ALG mic os uc u es we e u he embedded wi hin a suppo ing bioma e ial
consis ing o XG-GMA, which, a e pho oc osslinking and imme sion in an EDTA aqueous
solu ion, enabled enginee ing he hollow mic ochannels. Then, HUVECs we e seeded on o
he mic ochannels aiming o colonize hem and o m p e ascula ne wo ks (Figu e 1).
Biomolecules 2021, 11, x 8 o 17
Figu e 1. Schema ic illus a ion o he p ocedu e behind he ab ica ion o 3D pe usable cons uc s encompassing
bioins uc i e (CHT/ALG-RGD)6 mul ilaye s empla ed on lique ied ALG mic ochannels embedded in pho oc osslinkable
XG-GMA suppo ing hyd ogels.
3.1. Fab ica ion o 3D P in ed ALG Sac i icial Templa e S uc u es
Cus omizable 3D ALG sac i icial empla e s uc u es exhibi ing di e en sizes and
shapes, om simple o mo e complex s uc u es, ha e been designed by he CAD model
and 3D p in ed, e ealing he high e sa ili y impa ed by he 3D p in ing echnology
(Figu e 2). The 3D p in ed ALG s uc u es we e s ong enough o sus ain handling and
main ain hei shape, as shown in Figu e 2E,F, hus holding g ea p omise o he design
and de elopmen o mo e in ica e and pe sonalized a i icial biological a chi ec u es o
be used in a wide a ay o applica ion scena ios, including in con olled he apeu ics
deli e y, biosensing, issue enginee ing and egene a i e medicine s a egies.
Figu e 1.
Schema ic illus a ion o he p ocedu e behind he ab ica ion o 3D pe usable cons uc s
encompassing bioins uc i e (CHT/ALG-RGD)
6
mul ilaye s empla ed on lique ied ALG mic ochan-
nels embedded in pho oc osslinkable XG-GMA suppo ing hyd ogels.
Biomolecules 2021,11, 863 8 o 16
3.1. Fab ica ion o 3D P in ed ALG Sac i icial Templa e S uc u es
Cus omizable 3D ALG sac i icial empla e s uc u es exhibi ing di e en sizes and
shapes, om simple o mo e complex s uc u es, ha e been designed by he CAD model
and 3D p in ed, e ealing he high e sa ili y impa ed by he 3D p in ing echnology
(Figu e 2). The 3D p in ed ALG s uc u es we e s ong enough o sus ain handling and
main ain hei shape, as shown in Figu e 2E,F, hus holding g ea p omise o he design
and de elopmen o mo e in ica e and pe sonalized a i icial biological a chi ec u es o be
used in a wide a ay o applica ion scena ios, including in con olled he apeu ics deli e y,
biosensing, issue enginee ing and egene a i e medicine s a egies.
Biomolecules 2021, 11, x 9 o 17
Figu e 2. Op ical images o he 3D p in ed ALG sac i icial empla e s uc u es, exhibi ing hei di e en sizes and shapes,
a e c osslinking wi h CaCl
2
: (A) cylinde , (B) sinusoidal, (C) s a and (D) capilla y s uc u es. (E) S a and (F) capilla y
3D p in ed pho og aphs, showing hei handling abili y.
3.2. Build-Up o Bioins uc i e CHT/ALG-RGD Mul ilaye ed Thin Films on 2D and 3D
Su aces
P io o he LbL su ace unc ionaliza ion o he 2D qua z c ys al senso s and 3D
p in ed ALG- empla ed sac i icial s uc u es wi h bioins uc i e (CHT/ALG-RGD)
6
bilaye s, he ne elec ical cha ge o eshly p epa ed CHT and RGD-coupled ALG
aqueous solu ions a he wo king pH o 5.5 was assessed by measu ing hei ζ-po en ials.
The ζ-po en ials o ALG (co e empla e), ALG-RGD and CHT aqueous solu ions a pH 5.5
we e ound o be −23.4 ± 1.6 mV, −19.7 ± 2.7 mV and +17.9 ± 0.4 mV, espec i ely, hus
un eiling he anionic na u e o ALG and ALG-RGD (pH > pK
a
~3.38 o 3.65 o
mannu onic o gulu onic acid esidues, espec i ely) [51,61], and he ca ionic na u e o
he CHT biopolyme ic solu ions (pH < pK
a
~6–6.5) [62]. As such, we hypo hesize ha he
posi i ely cha ged CHT and he nega i ely cha ged ALG-RGD biopolyme s could
success ully build-up elec os a ic-d i en mul ilaye ed hin ilms by exploi ing he
a ac i e elec os a ic in e ac ions be ween he opposi ely cha ged biopolyme s. Hence,
he possible build-up o 2D CHT/ALG-RGD mul ilaye ed hin ilms ia elec os a ic
in e ac ions be ween he opposi ely cha ged biocompa ible CHT and RGD-coupled ALG
biopolyme s was assessed in si u by he QCM-D echnique by applying an al e na ing
elec ic ield ac oss he gold-coa ed qua z c ys al senso [63]. The QCM-D echnique
allows us o de ec e y small changes in he hyd odynamic mass (ng cm
−2
) due o changes
in he esonance equency o he qua z c ys al senso and measu e he iscoelas ic
p ope ies o he adso bed laye s h ough he ene gy dissipa ed in he mechanical
oscilla ion o he qua z senso s [63,64].
Figu e 3A showcases he no malized equency (Δ
n
/n) and dissipa ion ac o (ΔD
n
)
changes ob ained a he 3 d
(n = 3; 15 MHz), 5 h
(n = 5; 25 MHz), 7 h
(n = 7; 35 MHz), 9 h
(n
= 9; 45 MHz) and 11 h o e ones (n = 11; 55 MHz) du ing he build-up o mul ilaye ed hin
ilms, encompassing six CHT/ALG-RGD bilaye s, on o he gold-pla ed qua z c ys al
subs a e.
The sequen ial dec ease in he Δ
n
/n a e he adso p ion o each biopolyme ic
aqueous solu ion, i.e., CHT and ALG-RGD, on o he Au-pla ed qua z c ys al su ace
p o es he deposi ion and e ec i e in e ac ion o he deposi ed ma e ials h oughou he
deposi ion cycles.
Mo eo e , he successi e inc ease in he ΔD
n
a e each deposi ion s ep oge he wi h
he sepa a ion deno ed by he di e en o e ones, mainly o he la es adso bed bilaye s,
e eal he iscoelas ici y o he adso bed laye s, which a e no cons an h oughou he
Figu e 2.
Op ical images o he 3D p in ed ALG sac i icial empla e s uc u es, exhibi ing hei di e en sizes and shapes,
a e c osslinking wi h CaCl
2
: (
A
) cylinde , (
B
) sinusoidal, (
C
) s a and (
D
) capilla y s uc u es. (
E
) S a and (
F
) capilla y 3D
p in ed pho og aphs, showing hei handling abili y.
3.2. Build-Up o Bioins uc i e CHT/ALG-RGD Mul ilaye ed Thin Films on 2D and 3D Su aces
P io o he LbL su ace unc ionaliza ion o he 2D qua z c ys al senso s and 3D
p in ed ALG- empla ed sac i icial s uc u es wi h bioins uc i e (CHT/ALG-RGD)
6
bi-
laye s, he ne elec ical cha ge o eshly p epa ed CHT and RGD-coupled ALG aqueous
solu ions a he wo king pH o 5.5 was assessed by measu ing hei
ζ
-po en ials. The
ζ
-po en ials o ALG (co e empla e), ALG-RGD and CHT aqueous solu ions a pH 5.5
we e ound o be
−
23.4
±
1.6 mV,
−
19.7
±
2.7 mV and +17.9
±
0.4 mV, espec i ely, hus
un eiling he anionic na u e o ALG and ALG-RGD (pH > pK
a
~3.38 o 3.65 o man-
nu onic o gulu onic acid esidues, espec i ely) [
51
,
61
], and he ca ionic na u e o he CHT
biopolyme ic solu ions (pH < pK
a
~6–6.5) [
62
]. As such, we hypo hesize ha he posi i ely
cha ged CHT and he nega i ely cha ged ALG-RGD biopolyme s could success ully build-
up elec os a ic-d i en mul ilaye ed hin ilms by exploi ing he a ac i e elec os a ic
in e ac ions be ween he opposi ely cha ged biopolyme s. Hence, he possible build-up
o 2D CHT/ALG-RGD mul ilaye ed hin ilms ia elec os a ic in e ac ions be ween he
opposi ely cha ged biocompa ible CHT and RGD-coupled ALG biopolyme s was assessed
in si u by he QCM-D echnique by applying an al e na ing elec ic ield ac oss he gold-
coa ed qua z c ys al senso [
63
]. The QCM-D echnique allows us o de ec e y small
changes in he hyd odynamic mass (ng cm
−2
) due o changes in he esonance equency
o he qua z c ys al senso and measu e he iscoelas ic p ope ies o he adso bed laye s
h ough he ene gy dissipa ed in he mechanical oscilla ion o he qua z senso s [63,64].
Figu e 3A showcases he no malized equency (
∆
n
/n) and dissipa ion ac o (
∆
D
n
)
changes ob ained a he 3 d (n= 3; 15 MHz), 5 h (n= 5; 25 MHz), 7 h (n= 7; 35 MHz),
9 h (n= 9; 45 MHz) and 11 h o e ones (n= 11; 55 MHz) du ing he build-up o mul ilay-
Biomolecules 2021,11, 863 9 o 16
e ed hin ilms, encompassing six CHT/ALG-RGD bilaye s, on o he gold-pla ed qua z
c ys al subs a e.
Biomolecules 2021, 11, x 10 o 17
adso p ion p ocess. This is a ypical ea u e o so and hyd a ed polyme ic ilms [65]. In
o e all, he dec ease in Δ
n
/n and he inc ease in he ΔD
n
alues sugges ha he nega i ely
cha ged ALG-RGD molecules a e adso bed on o he posi i ely cha ged CHT laye ,
con i ming he elec os a ic-d i en in e ac ion be ween bo h biopolyme s and he s able
s ep-by-s ep g ow h o he LbL assemblies. In addi ion, he insing s eps did no induce
changes in he Δ
n
/n and ΔD
n
da a a e he deposi ion o each laye ed ma e ial, hus
con i ming he s ong in e ac ion be ween he biopolyme s, as well as he i e e sible
na u e o he adso p ion p ocess.
Figu e 3. Build-up o (CHT/ALG-RGD)
6
mul ilaye ed hin ilms on o Au-pla ed qua z c ys al senso s. (A) QCM-D
moni o ing o he no malized equency (Δ
n
/n) and dissipa ion (ΔD
n
) shi s, ob ained a he 3 d
(n = 3; 15 MHz), 5 h (n =
5; 25 MHz), 7 h
(n = 7; 35 MHz), 9 h
(n = 9; 45 MHz) and 11 h o e ones (n = 11; 55 MHz), as a unc ion o ime o he build-
up o (CHT/ALG-RGD)
6
bilaye s on o gold-pla ed qua z c ys al senso s and in e media e insing s eps. The numbe s
e e o he adso p ion o CHT (1), ALG-RGD (3) and insing s eps (2 and 4). (B) Cumula i e hyd odynamic hickness
e olu ion o he (CHT/ALG-RGD)
6
mul ilaye ed hin ilms, es ima ed using he Voig -based iscoelas ic model. The black
s aigh line ep esen s he linea eg ession i accompanied by he espec i e coe icien o de e mina ion (R
2
= 0.9915).
The hyd odynamic hickness o he mul ilaye ed ilm pe adso bed laye was also
es ima ed using he Voig -based iscoelas ic model [66], as showcased in Figu e 3B,
e ealing he nanos uc u ed dimension o he adso bed laye s. Fu he mo e, a linea
inc ease in he hyd odynamic hickness along he adso p ion cycles was obse ed,
co obo a ing he inc ease in he −Δ
n
/n and ΔD
n
alues. The build-up o he (CHT/ALG-
RGD)
6
mul ilaye ed hin ilm led o a inal mul ilaye ed hickness o ca. 58 nm.
Following he success ul in e ac ion and he s able LbL g ow h o he bioac i e
(CHT/ALG-RGD)
6
mul ilaye ed hin ilms on 2D qua z c ys al senso s, simila
mul ilaye ed hin ilms ha e been success ully and con o mally empla ed on 3D p in ed
ALG sac i icial mic o ibe empla es (ca. 1150 μm in diame e , co obo a ing a p e ious
epo [29]), using an au oma ic dipping obo , as con i med by labeling he CHT
biopolyme wi h RITC (Figu e 4). Al hough he e ec i e LbL coa ing has been
demons a ed o he 3D p in ed ALG ibe -like s uc u es, he high e sa ili y impa ed
by he LbL assembly echnology enables coa ing i ually any ype o empla e, ega dless
o i s size, shape, and su ace chemis y, hus opening new a enues in modula issue
enginee ing. P e iously, we ha e demons a ed such e sa ili y by coa ing ALG hyd ogel
pa icles wi h a simila mul ilaye ed hin coa ing [67–69]. In addi ion, we ha e
demons a ed ha a minimum o six bilaye s was needed o p oduce a mechanically
s able, obus , and con o mal co e-shell s uc u e ha could sus ain i s manipula ion and
implan a ion [68], hus being he a ionale behind coa ing he 3D p in ed ALG s uc u es
wi h six bilaye s. Mo eo e , we ha e also alida ed p e iously he hin mul ilaye ed
nanocoa ing as a pe mselec i e memb ane, enabling he di usion o essen ial molecules
o cell su i al, such as nu ien s and oxygen, and he exchange o me aboli es and was e
p oduc s.
Figu e 3.
Build-up o (CHT/ALG-RGD)
6
mul ilaye ed hin ilms on o Au-pla ed qua z c ys al senso s. (
A
) QCM-D
moni o ing o he no malized equency (
∆
n
/n) and dissipa ion (
∆
D
n
) shi s, ob ained a he 3 d (n= 3; 15 MHz), 5 h (n= 5;
25 MHz), 7 h (n= 7; 35 MHz), 9 h (n= 9; 45 MHz) and 11 h o e ones (n= 11; 55 MHz), as a unc ion o ime o he build-up
o (CHT/ALG-RGD)
6
bilaye s on o gold-pla ed qua z c ys al senso s and in e media e insing s eps. The numbe s e e o
he adso p ion o CHT (1), ALG-RGD (3) and insing s eps (2 and 4). (
B
) Cumula i e hyd odynamic hickness e olu ion o
he (CHT/ALG-RGD)
6
mul ilaye ed hin ilms, es ima ed using he Voig -based iscoelas ic model. The black s aigh line
ep esen s he linea eg ession i accompanied by he espec i e coe icien o de e mina ion (R2= 0.9915).
The sequen ial dec ease in he
∆
n
/n a e he adso p ion o each biopolyme ic aqueous
solu ion, i.e., CHT and ALG-RGD, on o he Au-pla ed qua z c ys al su ace p o es he depo-
si ion and e ec i e in e ac ion o he deposi ed ma e ials h oughou he
deposi ion cycles.
Mo eo e , he successi e inc ease in he
∆
D
n
a e each deposi ion s ep oge he wi h
he sepa a ion deno ed by he di e en o e ones, mainly o he la es adso bed bilaye s,
e eal he iscoelas ici y o he adso bed laye s, which a e no cons an h oughou he
adso p ion p ocess. This is a ypical ea u e o so and hyd a ed polyme ic ilms [
65
].
In o e all, he dec ease in
∆
n
/n and he inc ease in he
∆
D
n
alues sugges ha he
nega i ely cha ged ALG-RGD molecules a e adso bed on o he posi i ely cha ged CHT
laye , con i ming he elec os a ic-d i en in e ac ion be ween bo h biopolyme s and he
s able s ep-by-s ep g ow h o he LbL assemblies. In addi ion, he insing s eps did no
induce changes in he
∆
n
/n and
∆
D
n
da a a e he deposi ion o each laye ed ma e ial,
hus con i ming he s ong in e ac ion be ween he biopolyme s, as well as he i e e sible
na u e o he adso p ion p ocess.
The hyd odynamic hickness o he mul ilaye ed ilm pe adso bed laye was also es i-
ma ed using he Voig -based iscoelas ic model [
66
], as showcased in Figu e 3B, e ealing
he nanos uc u ed dimension o he adso bed laye s. Fu he mo e, a linea inc ease in he
hyd odynamic hickness along he adso p ion cycles was obse ed, co obo a ing he in-
c ease in he
−∆
n
/n and
∆
D
n
alues. The build-up o he (CHT/ALG-RGD)
6
mul ilaye ed
hin ilm led o a inal mul ilaye ed hickness o ca. 58 nm.
Following he success ul in e ac ion and he s able LbL g ow h o he bioac i e
(CHT/ALG-RGD)
6
mul ilaye ed hin ilms on 2D qua z c ys al senso s, simila mul-
ilaye ed hin ilms ha e been success ully and con o mally empla ed on 3D p in ed ALG
sac i icial mic o ibe empla es (ca. 1150
µ
m in diame e , co obo a ing a p e ious e-
po [
29
]), using an au oma ic dipping obo , as con i med by labeling he CHT biopolyme
wi h RITC (Figu e 4). Al hough he e ec i e LbL coa ing has been demons a ed o he
3D p in ed ALG ibe -like s uc u es, he high e sa ili y impa ed by he LbL assembly
echnology enables coa ing i ually any ype o empla e, ega dless o i s size, shape, and
su ace chemis y, hus opening new a enues in modula issue enginee ing. P e iously,
we ha e demons a ed such e sa ili y by coa ing ALG hyd ogel pa icles wi h a simila
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