Ci a ion: Conejo-Cue as, G.;
Ruiz-Rubio, L.; Sáez-Ma ínez, V.;
Pé ez-González, R.; Ga ziandia, O.;
Hugue -Casque o, A.; Pé ez-Ál a ez,
L. Spon aneous Gela ion o Adhesi e
Ca echol Modi ied Hyalu onic Acid
and Chi osan. Polyme s 2022,14, 1209.
h ps://doi.o g/10.3390/
polym14061209
Academic Edi o : Lumini a Ma in
Recei ed: 28 Janua y 2022
Accep ed: 14 Ma ch 2022
Published: 17 Ma ch 2022
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Licensee MDPI, Basel, Swi ze land.
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A ibu ion (CC BY) license (h ps://
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polyme s
A icle
Spon aneous Gela ion o Adhesi e Ca echol Modi ied
Hyalu onic Acid and Chi osan
Guille mo Conejo-Cue as 1, Lei e Ruiz-Rubio 1,2 , Vi ginia Sáez-Ma ínez 3, Raul Pé ez-González 3,
Oihane Ga ziandia 3, Amaia Hugue -Casque o 3and Ley e Pé ez-Ál a ez 1,2,*
1Mac omolecula Chemis y G oup (LABQUIMAC), Depa men o Physical Chemis y,
Facul y o Science and Technology, Uni e si y o he Basque Coun y, UPV/EHU, Ba io Sa iena, s/n,
48940 Leioa, Spain; [email p o ec ed] (G.C.-C.); lei e. [email p o ec ed] (L.R.-R.)
2BCMa e ials, Basque Cen e o Ma e ials, Applica ions and Nanos uc u es, UPV/EHU Science Pa k,
48940 Leioa, Spain
3i+Med S. Coop. Pa que Tecnológico de Ála a, Albe Eins ein 15, na e 15, 01510 Vi o ia-Gas eiz, Spain;
[email p o ec ed] (V.S.-M.); [email p o ec ed] (R.P.-G.); [email p o ec ed] (O.G.);
[email p o ec ed] (A.H.-C.)
*Co espondence: ley e.pe [email p o ec ed]
Abs ac :
Spon aneously o med hyd ogels a e a ac ing inc easing in e es as injec able o wound
d essing ma e ials because hey do no equi e addi ional eac ions o oxic c osslinking eagen s.
Highly aluable p ope ies such as low iscosi y be o e ex e nal applica ion, adequa e ilmogenic
capaci y, apid gela ion and issue adhesion a e equi ed in o de o use hem o hose he apeu ic
applica ions. In addi ion, biocompa ibili y and biodeg adabili y a e also manda o y. Acco dingly,
biopolyme s, such as hyalu onic acid (HA) and chi osan (CHI), ha ha e shown g ea po en ial
o wound healing applica ions a e excellen candida es due o hei unique physiochemical and
biological p ope ies, such as mois u izing and an imic obial abili y, espec i ely. In his s udy, bo h
biopolyme s we e modi ied by co alen ancho ing o ca echol g oups, and he ob ained hyd ogels
we e cha ac e ized by s udying, in pa icula , hei issue adhesi eness and ilm o ming capaci y o
po en ial skin wound healing applica ions. Tissue adhesi eness was ela ed o o-quinone o ma ion
o e ime and moni o ed by isible spec oscopy. Consequen ly, an opposi e e ec was obse ed o
bo h polysaccha ides. As gela ion ad ances o HA-CA, i becomes mo e adhesi e, while compe i i e
eac ions o quinone in CHI-CA slow down issue adhesi eness and induce a de imen o he
ilmogenic p ope ies.
Keywo ds: hyalu onic acid; chi osan; ca echol; issue adhesi e
1. In oduc ion
Hyd ogels a e polyme s based on h ee-dimensional ne wo ks capable o e aining
la ge amoun s o wa e due o hei hyd ophilic na u e, while emaining insoluble due
o polyme chains c osslinking [
1
]. C osslinking by co alen bonds esul s in co alen ly
c osslinked ne wo ks, and when polyme s a e joined by non-co alen in e ac ions such as
hyd ogen bonds and hyd ophobic o dipole–dipole in e ac ions, physical hyd ogels a e
o med [
2
]. D ied hyd ogels beha e simila ly o a ha d solid, bu in an aqueous medium,
wa e pene a ion be ween he polyme chains causes he swelling o he ne wo k [
3
,
4
].
Wa e con en a ec s d ama ically he mechanical p ope ies o hese gels [
1
], leading o so ,
elas ic and pe meable ma e ials o which i s p ope ies a e simila o hose o biological
issues [
5
]. Fo his eason, hyd ogels a e well known as in e es ing ma e ials in biomedical
applica ions [6].
Hyd ogel cha ac e is ics make hem in e es ing candida es o wound healing applica-
ions. On he one hand, hei hyd ophilic na u e allows he equi ed mois en i onmen
in he wound o ex acellula ma ix o ma ion and e-epi helializa ion and p o ides
Polyme s 2022,14, 1209. h ps://doi.o g/10.3390/polym14061209 h ps://www.mdpi.com/jou nal/polyme s
Polyme s 2022,14, 1209 2 o 16
p o ec ion agains in ec ions. On he o he hand, he inco po a ion o he apeu ic agen s
in o hyd ogels ac ing as wound d essings p o ides hei opical elease in he wound ha
has been shown o be mo e e ec i e han sys emic ea men [
7
]. Indeed, he p omo ion
o an e ec i e wound healing o egene a ion, which consis s o a se ies o complex bio-
chemical eac ions ha aims o epai he wound, is highly demanded. This p ocess occu s
in h ee s ages ha can ake place simul aneously. Fi s ly, he in lamma ion phase akes
place, which can be summa ized as he elimina ion o bac e ia and he mig a ion o cells
ha ac in he second s age. Secondly, he p oli e a ion phase comp ises an inc ease in
collagen wi h he aim o o ming new issues and blood essels, as well as he con ac ion
o he wound. Las ly, in he las phase called ma u a ion, he elimina ion o he excess
cells and he eposi ioning o he collagen occu . This en i e p ocess is complex and highly
suscep ible o be in e up ed o ail [
8
]. Due o he la e , his p ocess can be suppo ed
by healing species, which can help by unc ioning as an ibac e ial ba ie s o by ac ing as
cellula sca olds ha enhance wound closu e [6].
Hyd ophilic polyme s, due o hei abili y o mimic physical and biological p ope ies
o issues, can p omo e damaged issue egene a ion. In his sense, i is wo h highligh ing
ha hyd ogels a e de i ed om na u al polyme s, especially polysaccha ides, which
ha e been widely in es iga ed and exploi ed in ecen yea s due o hei abundance,
biocompa ible, ilmogenic, and bene icial biological p ope ies ha make hem in e es ing
candida es o wound d essing applica ions [
2
]. The mos s udied polysaccha ides include
algina es, chond oi in, chi osan and chi in, cellulose, dex an, hyalu onic acid and hepa in.
Hyalu onic acid (HA) is a na u al polysaccha ide based on a D-glucu onic acid and
N-ace yl-D-glucosamine (Figu e 1b) ha is used in a ious biomedical applica ions, such
as wound healing, isco-supplemen a ion o w inkle ille s, d ug deli e y ca ie s and
issue sca olds. Fu he mo e, his polysaccha ide is comple ely deg aded in he body
by hyalu onidase, in which he eloci y o biodeg ada ion is in luenced by i s molecula
weigh [
9
]. HA can in e ac in a/in e molecula ly hanks o hyd ogen bonds o ionic
in e ac ions by i s ca boxylic g oups and hei dep o ona ed o m, ca boxyla es. I also can
be easily modi ied by i s ca boxyl o hyd oxyl g oups. The e a e many examples o HA
modi ied hyd ogels o healing, such as hyalu onic acid modi ied wi h b omo ace a e o
hose modi ied wi h polyhyd azides [10].
Polyme s 2022, 14, x FOR PEER REVIEW 3 o 16
Figu e 1. (a) Molecula s uc u e o hyalu onic acid showing D-glucu onic acid (le ) and a N-
ace yl-D-glucosamine ( igh ) uni s. (b) Molecula s uc u e o chi osan monome s possessing D-
glucosamine (le ) and N-ace ylglucosamine ( igh ).
Due o he p esence o ci ed chemical g oups in i s s uc u e, CHI is e y good a
in e ac ing h ough hyd ogen bonds, and by elec os a ic in e ac ions wi h nega i e
cha ges a he app op ia e pH due o he p o ona ion o i s amino g oups (-NH
3+
) [2].
Since wound healing ea men equi es p olonged ime pe iods, he de elopmen
o ilmogenic ma e ials wi h issue adhesi eness, such as adhesi e hyd ogels, is c ucial
o a sui able pe o mance on he skin. Fo his, ac yla e de i ed hyd ogels ha e been
ypically de eloped in he las decades based on hei adhesi e p ope ies [11,15].
Tissue adhesion (Figu e 2) is p omo ed by he in e ac ion o issues wi h many unc-
ional g oups ha a e p esen along hyd ogels polyme ic chains h ough co alen bonds,
such as imine o ma ions o Schi bases and Michael addi ions, among o he s. Mo eo-
e , issue adhesion p omo ed by physical in e ac ions such as hyd ogen bonding is he
mos equen . Howe e , hese in e ac ions a e e e sible, which causes a dec ease in he
abili y o he hyd ogel o emain a ached o issues [16].
Figu e 2. In e molecula in e ac ions be ween polyme chains and issue.
In na u e, he adhesi e abili y o mussels has been asc ibed o he p esence o an
amino acid: L-3,4-dihyd oxyphenylalanine (DOPA), which is esponsible o hei adhe-
sion o bo h ino ganic and o ganic su aces, especially in humid condi ions. This dihy-
d oxy g oup is called ca echol (CA) [17,18].
Taking he inspi a ion o hese na u al o ganisms, he s a egy o modi ying poly-
me s wi h ca echol g oups has ecen ly been de eloped o disco e new ma e ials, such
as hyd ogels wi h adhesi e p ope ies [12]. The de i a i es o ca echol a e pa icula ly
in e es ing, as hey a e also na u al and, he e o e, biodeg adable and biocompa ible.
The main ad an age o na u al polysaccha ides chi osan and hyalu onic acid elies on
he ac ha hey a e easily modi iable h ough chemical eac ions by hei amine o
Figu e 1.
(
a
) Molecula s uc u e o hyalu onic acid showing D-glucu onic acid (le ) and a N-ace yl-
D-glucosamine ( igh ) uni s. (
b
) Molecula s uc u e o chi osan monome s possessing D-glucosamine
(le ) and N-ace ylglucosamine ( igh ).
Polyme s 2022,14, 1209 3 o 16
Chi osan (CHI) is also a na u al polyme ha comes om he pa ial deace yla ion o
chi in, a na u al polyme syn hesized by some a h opods, ungi and insec s [
11
,
12
]. Thus,
chi osan has a D-glucosamine s uc u e mixed wi h N-ace ylglucosamine s uc u es o he
ace yla ed monome , as obse ed in Figu e 1a. Chi osan is deg aded in he human body
by he ac ion o lysozyme and colonic bac e ial enzymes and i s biodeg ada ion s ongly
depends on i s deace yla ion deg ee and molecula weigh [
2
]. I is conside ed one o
he mos p omising ma e ials in he ields o pha macy, chemis y and he ood indus y
due o i s highly eac i e hyd oxyl and amino g oups, as well as being a biocompa ible,
an ibac e ial and non oxic polyme [
13
]. I is also no ewo hy i s abili y o o m ilms,
which i is able o cause he supp ession o essen ial nu ien s o mic obial g ow h, in o he
wo ds p o ec ing he open wound om he ou side due o i s good ba ie p ope ies [
14
].
Due o he p esence o ci ed chemical g oups in i s s uc u e, CHI is e y good a
in e ac ing h ough hyd ogen bonds, and by elec os a ic in e ac ions wi h nega i e cha ges
a he app op ia e pH due o he p o ona ion o i s amino g oups (-NH3+) [2].
Since wound healing ea men equi es p olonged ime pe iods, he de elopmen o
ilmogenic ma e ials wi h issue adhesi eness, such as adhesi e hyd ogels, is c ucial o a
sui able pe o mance on he skin. Fo his, ac yla e de i ed hyd ogels ha e been ypically
de eloped in he las decades based on hei adhesi e p ope ies [11,15].
Tissue adhesion (Figu e 2) is p omo ed by he in e ac ion o issues wi h many unc-
ional g oups ha a e p esen along hyd ogels polyme ic chains h ough co alen bonds,
such as imine o ma ions o Schi bases and Michael addi ions, among o he s. Mo eo e ,
issue adhesion p omo ed by physical in e ac ions such as hyd ogen bonding is he mos
equen . Howe e , hese in e ac ions a e e e sible, which causes a dec ease in he abili y
o he hyd ogel o emain a ached o issues [16].
Polyme s 2022, 14, x FOR PEER REVIEW 3 o 16
Figu e 1. (a) Molecula s uc u e o hyalu onic acid showing D-glucu onic acid (le ) and a N-
ace yl-D-glucosamine ( igh ) uni s. (b) Molecula s uc u e o chi osan monome s possessing D-
glucosamine (le ) and N-ace ylglucosamine ( igh ).
Due o he p esence o ci ed chemical g oups in i s s uc u e, CHI is e y good a
in e ac ing h ough hyd ogen bonds, and by elec os a ic in e ac ions wi h nega i e
cha ges a he app op ia e pH due o he p o ona ion o i s amino g oups (-NH
3+
) [2].
Since wound healing ea men equi es p olonged ime pe iods, he de elopmen
o ilmogenic ma e ials wi h issue adhesi eness, such as adhesi e hyd ogels, is c ucial
o a sui able pe o mance on he skin. Fo his, ac yla e de i ed hyd ogels ha e been
ypically de eloped in he las decades based on hei adhesi e p ope ies [11,15].
Tissue adhesion (Figu e 2) is p omo ed by he in e ac ion o issues wi h many unc-
ional g oups ha a e p esen along hyd ogels polyme ic chains h ough co alen bonds,
such as imine o ma ions o Schi bases and Michael addi ions, among o he s. Mo eo-
e , issue adhesion p omo ed by physical in e ac ions such as hyd ogen bonding is he
mos equen . Howe e , hese in e ac ions a e e e sible, which causes a dec ease in he
abili y o he hyd ogel o emain a ached o issues [16].
Figu e 2. In e molecula in e ac ions be ween polyme chains and issue.
In na u e, he adhesi e abili y o mussels has been asc ibed o he p esence o an
amino acid: L-3,4-dihyd oxyphenylalanine (DOPA), which is esponsible o hei adhe-
sion o bo h ino ganic and o ganic su aces, especially in humid condi ions. This dihy-
d oxy g oup is called ca echol (CA) [17,18].
Taking he inspi a ion o hese na u al o ganisms, he s a egy o modi ying poly-
me s wi h ca echol g oups has ecen ly been de eloped o disco e new ma e ials, such
as hyd ogels wi h adhesi e p ope ies [12]. The de i a i es o ca echol a e pa icula ly
in e es ing, as hey a e also na u al and, he e o e, biodeg adable and biocompa ible.
The main ad an age o na u al polysaccha ides chi osan and hyalu onic acid elies on
he ac ha hey a e easily modi iable h ough chemical eac ions by hei amine o
Figu e 2. In e molecula in e ac ions be ween polyme chains and issue.
In na u e, he adhesi e abili y o mussels has been asc ibed o he p esence o an amino
acid: L-3,4-dihyd oxyphenylalanine (DOPA), which is esponsible o hei adhesion o bo h
ino ganic and o ganic su aces, especially in humid condi ions. This dihyd oxy g oup is
called ca echol (CA) [17,18].
Taking he inspi a ion o hese na u al o ganisms, he s a egy o modi ying polyme s
wi h ca echol g oups has ecen ly been de eloped o disco e new ma e ials, such as
hyd ogels wi h adhesi e p ope ies [
12
]. The de i a i es o ca echol a e pa icula ly
in e es ing, as hey a e also na u al and, he e o e, biodeg adable and biocompa ible. The
main ad an age o na u al polysaccha ides chi osan and hyalu onic acid elies on he ac
ha hey a e easily modi iable h ough chemical eac ions by hei amine o ca boxylic
acid g oups, espec i ely [
9
,
19
]. Mo eo e , he CA g oup can be oxidized a basic pH
o e en in he p esence o he oxygen o he a mosphe e [
9
], and i is ans o med o o-
quinone (Figu e 3a). I can also be oxidized in en ionally and a a highe a e wi h sodium
pe ioda e [
20
]. This spon aneously o med g oup beha es as a Michael accep o and eac s
wi h speci ic subs a es, such as amines, hiols, alcohols, e c. [21,22].
Polyme s 2022,14, 1209 4 o 16
Polyme s 2022, 14, x FOR PEER REVIEW 4 o 16
ca boxylic acid g oups, espec i ely [9,19]. Mo eo e , he CA g oup can be oxidized a
basic pH o e en in he p esence o he oxygen o he a mosphe e [9], and i is ans-
o med o o-quinone (Figu e 3a). I can also be oxidized in en ionally and a a highe a e
wi h sodium pe ioda e [20]. This spon aneously o med g oup beha es as a Michael ac-
cep o and eac s wi h speci ic subs a es, such as amines, hiols, alcohols, e c. [21,22].
Figu e 3. (a) Ca echol g oup oxida ion o quinone by a mosphe ic oxygen. (b) C osslinking be-
ween ca echol and quinone g oups. R equals o CHI o HA.
In he case o o ganic su aces ha con ain elec on dono g oups such as alcohols,
hiols o amines, quinone eac s i e e sibly, making mo e esis an co alen bonds han
physical in e ac ions o ca echol [23]. Once CA is oxidized o quinone, he polyme ha
ca ies his subs i uen begins o eac wi h i sel (Figu e 4) [22,23], causing i s sel -c oss-
linking, inc easing iscosi y, while a change o colou akes places and becomes b own-
ish. This eac ion esul s in a apid ha dening o he p oduc (Figu e 3b) ha can be seen
as an ad an age, because i is a me hod o spon aneously p omo ing he gela ion o he
polyme , which p oceeds om a iscous liquid s a e o a gelled s a e wi hou he addi-
ion o any ex e nal c osslinking agen s. Howe e , o he bes o ou knowledge, he e -
ec o his spon aneous gela ion on he issue adhesi eness and ilmogenic p ope ies o
hese polysaccha ides has no been explo ed and compa a i ely analyzed.
Figu e 4. HA-CA syn hesis eac ion a pH = 4–5 a oom empe a u e and unde ni ogen a mos-
phe e.
Taking all his in o accoun , his wo k aims o explo e he o ma ion o hyd ogels
o ca echol de i a i es ob ained by he chemical modi ica ion o chi osan and hyalu onic
acid as issue adhesi e and ilmogenic ma e ials o po en ial wound healing pu poses.
Figu e 3.
(
a
) Ca echol g oup oxida ion o quinone by a mosphe ic oxygen. (
b
) C osslinking be ween
ca echol and quinone g oups. R equals o CHI o HA.
In he case o o ganic su aces ha con ain elec on dono g oups such as alcohols,
hiols o amines, quinone eac s i e e sibly, making mo e esis an co alen bonds han
physical in e ac ions o ca echol [
23
]. Once CA is oxidized o quinone, he polyme ha ca -
ies his subs i uen begins o eac wi h i sel (Figu e 4) [
22
,
23
], causing i s sel -c osslinking,
inc easing iscosi y, while a change o colou akes places and becomes b ownish. This eac-
ion esul s in a apid ha dening o he p oduc (Figu e 3b) ha can be seen as an ad an age,
because i is a me hod o spon aneously p omo ing he gela ion o he polyme , which
p oceeds om a iscous liquid s a e o a gelled s a e wi hou he addi ion o any ex e nal
c osslinking agen s. Howe e , o he bes o ou knowledge, he e ec o his spon aneous
gela ion on he issue adhesi eness and ilmogenic p ope ies o hese polysaccha ides has
no been explo ed and compa a i ely analyzed.
Polyme s 2022, 14, x FOR PEER REVIEW 4 o 16
ca boxylic acid g oups, espec i ely [9,19]. Mo eo e , he CA g oup can be oxidized a
basic pH o e en in he p esence o he oxygen o he a mosphe e [9], and i is ans-
o med o o-quinone (Figu e 3a). I can also be oxidized in en ionally and a a highe a e
wi h sodium pe ioda e [20]. This spon aneously o med g oup beha es as a Michael ac-
cep o and eac s wi h speci ic subs a es, such as amines, hiols, alcohols, e c. [21,22].
Figu e 3. (a) Ca echol g oup oxida ion o quinone by a mosphe ic oxygen. (b) C osslinking be-
ween ca echol and quinone g oups. R equals o CHI o HA.
In he case o o ganic su aces ha con ain elec on dono g oups such as alcohols,
hiols o amines, quinone eac s i e e sibly, making mo e esis an co alen bonds han
physical in e ac ions o ca echol [23]. Once CA is oxidized o quinone, he polyme ha
ca ies his subs i uen begins o eac wi h i sel (Figu e 4) [22,23], causing i s sel -c oss-
linking, inc easing iscosi y, while a change o colou akes places and becomes b own-
ish. This eac ion esul s in a apid ha dening o he p oduc (Figu e 3b) ha can be seen
as an ad an age, because i is a me hod o spon aneously p omo ing he gela ion o he
polyme , which p oceeds om a iscous liquid s a e o a gelled s a e wi hou he addi-
ion o any ex e nal c osslinking agen s. Howe e , o he bes o ou knowledge, he e -
ec o his spon aneous gela ion on he issue adhesi eness and ilmogenic p ope ies o
hese polysaccha ides has no been explo ed and compa a i ely analyzed.
Figu e 4. HA-CA syn hesis eac ion a pH = 4–5 a oom empe a u e and unde ni ogen a mos-
phe e.
Taking all his in o accoun , his wo k aims o explo e he o ma ion o hyd ogels
o ca echol de i a i es ob ained by he chemical modi ica ion o chi osan and hyalu onic
acid as issue adhesi e and ilmogenic ma e ials o po en ial wound healing pu poses.
Figu e 4.
HA-CA syn hesis eac ion a pH = 4–5 a oom empe a u e and unde ni ogen a mosphe e.
Taking all his in o accoun , his wo k aims o explo e he o ma ion o hyd ogels o
ca echol de i a i es ob ained by he chemical modi ica ion o chi osan and hyalu onic acid
as issue adhesi e and ilmogenic ma e ials o po en ial wound healing pu poses.
2. Ma e ials and Me hods
2.1. Ma e ials
Fo he syn hesis o he hyd ogels, chi osan (CHI, 1.2
×
10
6±
153.9 g/mol, Sigma-
Ald ich, S . Louis, MO, USA; DD = 80%), hyalu onic acid (HA, 1.9–2.2
×
10
6
g/mol, Con-
ipo, DolníDob ouˇc, Czech Republic), hyd ochlo ic acid (HCl, 37%, Pan eac, Ba celona,
Polyme s 2022,14, 1209 5 o 16
Spain) and e hanol (E OH, 99.8%, Pan eac, Ba celona, Spain) as sol en we e used. 3,4-
Dihyd oxycinnamic acid o hyd oca eic acid (HCF, 98%, Sigma-Ald ich, S . Louis, MO,
USA) and dopamine hyd ochlo ide (DOPA, 98%, Sigma-Ald ich, S . Louis, MO, USA) we e
employed o in oduce he ca echol g oup. To ca y ou he conjuga ion o he ca echol
o he polyme , N-hyd oxysuccinimide (NHS, 98%, Sigma-Ald ich), S . Louis, MO, USA
and N-(3-Dime hylaminop opyl)-N
0
-e hylca bodiimide hyd ochlo ide (EDC, 98%, Sigma-
Ald ich, S . Louis, MO, USA) we e used. Subsequen ly, o clean he modi ied polyme , a
dialysis was ca ied ou wi h 12,000 Da memb anes (Medicell Memb anes L d., London,
UK). The magnesium chlo ide sal (MgCl
2
, 98%, Sigma-Ald ich) was used o con ol hu-
midi y in a closed a mosphe e. Sodium me ape ioda e (NaIO
4
, 99%, Sigma-Ald ich, S .
Louis, MO, USA) was used o ca echol oxida ion in spec oscopy calib a ion. In o de o
p epa e a phospha e bu e saline o PBS, monobasic sodium phospha e (NaH
2
PO
4
, 99%,
Sigma-Ald ich, S . Louis, MO, USA) and sodium hyd oxide (NaOH, 99%, Pan eac) we e
used. PET ilms (75 µm) we e supplied by HIFI Film Indus ia (S e enage, UK).
2.2. Expe imen al Syn hesis
2.2.1. Syn hesis o Hyalu onic Acid-Ca echol (HA-CA)
The syn hesis o hyalu onic acid wi h ca echol was ca ied ou ollowing he desc ibed
me hod [
24
]. B ie ly, high molecula weigh hyalu onic acid (1 g, 2.5 mmol) was dissol ed
in dis illed wa e (200 mL) o 12 h and unde a ni ogen a mosphe e. EDC (959 mg and
5 mmol
) and NHS (575 mg and 5 mmol) (Figu e 4) we e hen slowly added o he eac ion
lask. A e 20 min unde s i ing, dopamine hyd ochlo ide (948 mg, 5 mmol) was added
a pH 4–5 o 4 h. I was le o eac o e nigh and dialyzed in 12,000–14,000 Da dyalisis
memb anes agains acidi ied deionized wa e (pH 5) o 3 days. Finally, he p oduc was
lyophilized and s o ed in a acuum desicca o a 3 ◦C.
2.2.2. Chi osan-Ca echol Syn hesis
The syn hesis o chi osan modi ied wi h he ca echol g oup was ca ied ou ollowing
he desc ibed me hod [
17
]. B ie ly, high molecula weigh chi osan (591 mg and 1.6 mmol)
was dissol ed in 22.5 mL o wa e oge he wi h 2.5 mL o 1 M HCl o e nigh unde a
ni ogen a mosphe e. The nex day, hyd oca eic acid (600 mg and 3.25 mmol), p e iously
dissol ed in 1.5 mL in dis illed wa e , was added. Then, EDC (930 mg and 4.75 mmol)
and NHS (558 mg and 4.75 mmol) (Figu e 5), dissol ed in 50 mL o an e hanol/wa e
solu ion (1:1, / ), we e added. The eac ion was le o e nigh , and he pH alue was
be ween 4 and 5. The p oduc was dialyzed on 12,000–14,000 Da memb anes in acidi ied
deionized wa e (pH 5) o 3 days. Finally, he p oduc was lyophilized (Bench op F eeze
D ye ope a ing a −50 ◦C, 0.1 mBa ) and s o ed in a acuum desicca o a 3 ◦C.
Polyme s 2022, 14, x FOR PEER REVIEW 5 o 16
2. Ma e ials and Me hods
2.1. Ma e ials
Fo he syn hesis o he hyd ogels, chi osan (CHI, 1.2 × 10
6
± 153.9 g/mol, Sigma-
Ald ich, S . Louis, MO, USA; DD = 80%), hyalu onic acid (HA, 1.9–2.2 × 10
6
g/mol, Con-
ipo, Dolní Dob ouč, Czech Republic), hyd ochlo ic acid (HCl, 37%, Pan eac, Ba celona,
Spain) and e hanol (E OH, 99.8%, Pan eac, Ba celona, Spain) as sol en we e used. 3,4-
Dihyd oxycinnamic acid o hyd oca eic acid (HCF, 98%, Sigma-Ald ich, S . Louis, MO,
USA) and dopamine hyd ochlo ide (DOPA, 98%, Sigma-Ald ich, S . Louis, MO, USA)
we e employed o in oduce he ca echol g oup. To ca y ou he conjuga ion o he ca -
echol o he polyme , N-hyd oxysuccinimide (NHS, 98%, Sigma-Ald ich), S . Louis, MO,
USA and N-(3-Dime hylaminop opyl)-N′-e hylca bodiimide hyd ochlo ide (EDC, 98%,
Sigma-Ald ich, S . Louis, MO, USA) we e used. Subsequen ly, o clean he modi ied pol-
yme , a dialysis was ca ied ou wi h 12,000 Da memb anes (Medicell Memb anes L d.
London, UK). The magnesium chlo ide sal (MgCl
2
, 98%, Sigma-Ald ich) was used o
con ol humidi y in a closed a mosphe e. Sodium me ape ioda e (NaIO
4
, 99%, Sigma-
Ald ich, S . Louis, MO, USA) was used o ca echol oxida ion in spec oscopy calib a-
ion. In o de o p epa e a phospha e bu e saline o PBS, monobasic sodium phospha e
(NaH
2
PO
4
, 99%, Sigma-Ald ich, S . Louis, MO, USA) and sodium hyd oxide (NaOH,
99%, Pan eac) we e used. PET ilms (75 μm) we e supplied by HIFI Film Indus ia (S e-
enage, UK).
2.2. Expe imen al Syn hesis
2.2.1. Syn hesis o Hyalu onic Acid-Ca echol (HA-CA)
The syn hesis o hyalu onic acid wi h ca echol was ca ied ou ollowing he de-
sc ibed me hod [24]. B ie ly, high molecula weigh hyalu onic acid (1 g, 2.5 mmol) was
dissol ed in dis illed wa e (200 mL) o 12 h and unde a ni ogen a mosphe e. EDC
(959 mg and 5 mmol) and NHS (575 mg and 5 mmol) (Figu e 4) we e hen slowly added
o he eac ion lask. A e 20 min unde s i ing, dopamine hyd ochlo ide (948 mg, 5
mmol) was added a pH 4–5 o 4 h. I was le o eac o e nigh and dialyzed in 12,000–
14,000 Da dyalisis memb anes agains acidi ied deionized wa e (pH 5) o 3 days. Fi-
nally, he p oduc was lyophilized and s o ed in a acuum desicca o a 3 °C.
2.2.2. Chi osan-Ca echol Syn hesis
The syn hesis o chi osan modi ied wi h he ca echol g oup was ca ied ou ollow-
ing he desc ibed me hod [17]. B ie ly, high molecula weigh chi osan (591 mg and 1.6
mmol) was dissol ed in 22.5 mL o wa e oge he wi h 2.5 mL o 1 M HCl o e nigh
unde a ni ogen a mosphe e. The nex day, hyd oca eic acid (600 mg and 3.25 mmol),
p e iously dissol ed in 1.5 mL in dis illed wa e , was added. Then, EDC (930 mg and
4.75 mmol) and NHS (558 mg and 4.75 mmol) (Figu e 5), dissol ed in 50 mL o an e ha-
nol/wa e solu ion (1:1, / ), we e added. The eac ion was le o e nigh , and he pH
alue was be ween 4 and 5. The p oduc was dialyzed on 12,000–14,000 Da memb anes
in acidi ied deionized wa e (pH 5) o 3 days. Finally, he p oduc was lyophilized
(Bench op F eeze D ye ope a ing a −50 °C, 0.1 mBa ) and s o ed in a acuum desicca o
a 3 °C.
Figu e 5. CHI-CA syn hesis eac ion a pH = 4–5 a oom empe a u e unde ni ogen a mosphe e.
2.2.3. Films
HA-CA and CHI-CA ilms we e p epa ed by using a doc o blade echnique o o m
we ilms wi h well-de ined hicknesses om solu ions a a concen a ion o 7 g/L in wa e
a oom empe a u e. Films ha we e 1-millime e - hick we e ob ained on o he PET shee .
Polyme s 2022,14, 1209 6 o 16
2.3. Cha ac e iza ion Techniques
P o on nuclea magne ic esonance (
1
H NMR) spec a we e pe o med a oom em-
pe a u e on a B uke AV-500 spec ome e (500 MHz o 1H), using deu e a ed ace ic acid
and wa e as sol en s. Chemical shi s (
δ
) a e exp essed in pa s pe million wi h espec
o deu e a ed wa e . The concen a ion o quinone g oup was de e mined by ul a iole
and isible spec oscopy (UV-VIS) measu ing he abso bance a 414 nm, espec i ely, in
he Double beam Cin a303 GBC equipmen . The gela ion ime o p epa ed hyd ogels was
de e mined a di e en polysaccha ide concen a ions by he known in e ed ube es [
9
],
in which i is conside ed ha he gela ion poin co esponds o he momen in which he
solu ion s ops lowing once in e ing he ube. An in e ed op ical mic oscope Olympus
IX71 om Japan was used as a non-des uc i e echnique. Pho og aphs we e ob ained in
o de o s udy he s abili y o he ilms du ing and a e d ying. A Hi achi S-4800 b and
scanning elec on mic oscope (FEG-SEM) om Japan was used in o de o ob ain high
esolu ion images o he ilms a mic on scale. In he case o polyme s, a laye o gold
was applied o allow he mobili y o he elec ons because hey a e no conduc i e. The
adhesion o he syn hesized hyd ogels was de e mined by measu ing he o ce necessa y o
de ach gels om a piece o issue wi h mechanical es equipmen (Me o ec, MTE ), using
a 20 N load cell. Fo his pu pose, po cine skin wi hou ex e nal a was cu in o ci cula
sec ions o 196 mm
2
and kep o 4–5 h in a PBS solu ion (pH
≈
7.4) a 37
◦
C o simula e
physiological condi ions [
15
,
25
]. Then, skin was ixed wi h cyanoac yla e (Loc i e
®
) [
26
] o
a es ube and placed on he su ace o he gel sample. Finally, he o ce pe a ea equi ed
o de ach i om he sample was measu ed. The s ess–displacemen cu es we e ob ained
o each sample. All measu emen s we e conduc ed wi h he ollowing pa ame e s: es
speed: 2 mm/min; skin/sample con ac ime: 1 min; con ac a ea: 196 mm
2
; p eload: 0 N;
d op: 100%.
3. Resul s
3.1. Ca echol Conjuga ion
CHI and HA we e chemically modi ied, as desc ibed in he Expe imen al Sec ion, in
o de o in oduce ca echol g oups along polysaccha ides chains o p omo e gela ion and
enhance adhesi eness o biological issue. This conjuga ion was con i med and quan i ied
by
1
H NMR and UV analyses. Figu e 6compa es he
1
H-NMR spec a o ini ial HA,
dopamine hyd ochlo ide eagen and he inally modi ied HA-CA.
1
H-NMR Hyalu onic acid (D
2
O, 500 MHz, 20
◦
C):
δ
(ppm) = 4.30 (s, 2H, anome ic
CH), 3.00–4.00 (m, 10H, ing CH and CH2), 1.99 (s, 3H, ace amide).
1
H-NMR Dopamine Hyd ochlo ide (D
2
O, 500 MHz, 20
◦
C):
δ
(ppm) = 6.70 (m, 3H,
CH a oma ic ing), 3.15 (d, 2H, -CH2N), 2.75 (d, 2H, -CH2A ).
1
H-NMR Hyalu onic acid-ca echol (D
2
O, 500 MHz, 20
◦
C):
δ
(ppm) = 6.75–7.30 (m,
3H, CH a oma ic ing), 4.30 (s, 4H, anome ic CH), 3.00–4.00 (m, 20H, ing CH and CH
2
),
2.82 (d, 2H, CH2N), 2.80 (d, 2H, -CH2A ), 1.99 (s, 6H, ace amide).
The appea ance o new peaks co esponding o he phenyl hyd ogens o ca echol
moie ies (Figu e 6c) a 6.5–6.75 ppm and hose appea ing a 2.75 ppm asc ibed o he
alipha ic ca bons o ca echol [
9
] demons a es he success ul conjuga ion o HA wi h
ca echol unc ionali y. In addi ion, he in eg a ion o he peaks a 6.5–6.75 ppm wi h
espec o 1.99 ppm peak co esponding o he me hyl p o ons o he ace amide g oup o
HA allows he quan i ica ion o he pe cen age o in oduced ca echol g oups, ob aining
a e age subs i u ion alues o 38 ±8%.
Polyme s 2022,14, 1209 7 o 16
Polyme s 2022, 14, x FOR PEER REVIEW 7 o 16
Figu e 6. (a) High molecula weigh hyalu onic acid spec um. (b) Dopamine hyd ochlo ide spec-
um, a eagen ha adds ca echol o he p oduc . (c) Hyalu onic acid modi ied wi h ca echol (HA-
CA) spec um.
1
H-NMR Hyalu onic acid (D
2
O, 500 MHz, 20 °C): δ (ppm) = 4.30 (s, 2H, anome ic
CH), 3.00–4.00 (m, 10H, ing CH and CH
2
), 1.99 (s, 3H, ace amide).
Figu e 6.
(
a
) High molecula weigh hyalu onic acid spec um. (
b
) Dopamine hyd ochlo ide spec-
um, a eagen ha adds ca echol o he p oduc . (
c
) Hyalu onic acid modi ied wi h ca echol
(HA-CA) spec um.
Polyme s 2022,14, 1209 8 o 16
In he CH-CA spec um (Figu e 7c), he appea ance o he cha ac e is ic peaks o
ca echol g oups (6.5–6.75 ppm) can be obse ed, indica ing ha he eac ion akes place
success ully. In addi ion, he peaks a 2.5 ppm co esponding o he alipha ic ca bons o
hyd oca eic acid and he appea ance o he signal a 4 ppm, which co esponds o he
hyd ogen o he C2 o he glucosamine uni o he chi osan bound o ca echol, we e also
obse ed. The pe cen age o subs i u ion o ca echol was calcula ed by he in eg a ion o he
peak a 6.5–6.75 ppm wi h espec o ha o chi osan appea ing a 1.99 ppm, knowing he
deg ee o deace yla ion. Di e en syn hesis condi ions we e explo ed in CHI modi ica ion.
On he one hand, he ollowing esul s we e ob ained: CHI-CA 1 wi h 24 h o eac ion and
1:2 CHI:HCF eed a io, CHI-CA 2 wi h 8 h o eac ion and 1:2 CHI:HCF eed a io and,
inally, CHI-CA 3 wi h 12 h o eac ion and 1:1 CHI:HC eed a io. The esul ing pe cen age
o ca echol a ied acco ding o hese syn he ic condi ions (Table 1). Indeed, lowe eagen
equi alen s (CHI-CA 3) and eac ion ime (CHI-CA 2) esul ed in a signi ican dec ease in
he conjuga ion wi h ca echol.
Table 1. Subs i u ion pe cen ages o ca echol in he samples.
Sample Ca echol % (1H NMR) a
HA-CA 38 ±8
CHI-CA 1 82 ±10
CHI-CA 2 8 ±2
CHI-CA 3 2 ±2
an= 3.
1H-NMR Chi osan (D2O, 500 MHz, 20
◦
C):
δ
(ppm) = 4.50 (s, 2H, anome ic CH),
3.30–4.00 (m, 10H, ing CH and CH2), 3.10 (s, 2H, CH -N ing), 1.99 (s, 3H, ace amide).
1H-NMR Hyd oca eic acid (D2O, 500 MHz, 20
◦
C):
δ
(ppm) = 6.5–6.75 (m, 3H, CH o
he a oma ic ing), 2.60 (d, 2H, CH2COOH), 2.50 (d, 2H, CH2A ).
1H-NMR Chi osan-ca echol (D2O, 500 MHz, 20
◦
C):
δ
(ppm) = 6.5–6.75 (m, 3H, CH o
he a oma ic ing), 4.50 (s, 3H, anome ic CH), 4.20 (dd, 1H, CH-N (ca echol)), 3.25–3.80 (m,
15H, ing CH and CH2), 3.10 (s, 2H, CH-N), 2.60 (d, 2H, CH2COOH), 2.30 (d, 2H, CH2A ),
1.99 (s, 3H, ace amide).
3.2. Hyd ogel Fo ma ion o Ca echol De i a i es
The spon aneous oxida ion o he ca echol g oup leads o hei ans o ma ion o he
quinone g oup ha p esen s an abso p ion in he isible spec um a
λ
= 380–480 nm (de-
pending on he deg ee o oxida ion) [
27
]. Acco dingly, he quan i ica ion o quinone moie y
was ca ied ou by using he calib a ion cu e ob ained wi h a s anda d solu ion o 1 mM
dopamine hyd ochlo ide p e iously oxidized wi h sodium pe ioda e (1:1 Dopa/Pe ioda e).
When pe ioda e was added o he dopamine solu ion, i immedia ely ook on a yellow hue,
and a e 10 min, i became eddish and b own, since he abso p ion spec um a ies wi h
oxida ion ime. Fo his eason, he calib a ion was ca ied ou a he isosbes ic poin , a
which abso p ion is no a unc ion o ime [
27
] (
λ
= 413.6 nm, Abs = 1112C + 0.009R
2
= 0.991).
This colo change allows moni o ing he oxida ion o ca echol-modi ied polysaccha ides.
Polyme s 2022,14, 1209 9 o 16
Polyme s 2022, 14, x FOR PEER REVIEW 9 o 16
Figu e 7. (a) High molecula weigh chi osan spec um. (b) Hyd oca eic acid spec um. (c) Chi-
osan modi ied wi h ca echol (CHI-CA) spec um.
Figu e 7.
(
a
) High molecula weigh chi osan spec um. (
b
) Hyd oca eic acid spec um. (
c
) Chi osan
modi ied wi h ca echol (CHI-CA) spec um.
Polyme s 2022,14, 1209 16 o 16
28.
Vo olakos, K.; Isaye a, I.S.; do Luu, H.M.; Pa wa dhan, D.V.; Pollack, S.K. Ionically c oss-linked hyalu onic acid: We ing,
lub ica ion, and iscoelas ici y o a modi ied adhesion ba ie gel. Med. De ices E id. Res. 2011,4, 1. [C ossRe ] [PubMed]
29.
Smejkalo a, D.; Hue a-Angeles, G.; Ehlo a, T. Hyalu onan (Hyalu onic Acid): A Na u al Mois u ize o Skin Ca e. In Ha y’s
Cosme icology, 9 h ed.; Chemical Publishing Company: Glouces e , MA, USA, 2015; Volume 2, pp. 605–622.
30.
Olejnik, A.; Goscianska, J.; Zielinska, A.; Nowak, I. S abili y de e mina ion o he o mula ions con aining hyalu onic acid. In . J.
Cosme . Sci. 2015,37, 401–407. [C ossRe ]
31.
Lim, S.T.; Ma in, G.P.; Be y, D.J.; B own, M.B. P epa a ion and e alua ion o he
in i o
d ug elease p ope ies and mucoadhe-
sion o no el mic osphe es o hyalu onic acid and chi osan. J. Con ol. Release 2000,66, 281–292. [C ossRe ]
32.
Cao, W.; Yan, J.; Liu, C.; Zhang, J.; Wang, H.; Gao, X.; Yan, H.; Niu, B.; Li, W. P epa a ion and cha ac e iza ion o ca echol-g a ed
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