pha maceu ics
A icle
De elopmen and Cha ac e iza ion o Xan han Gum and
Algina e Based Bioadhesi e Film o Pycnogenol Topical Use in
Wound T ea men
Cinzia Pagano 1,* , Debo a Puglia 2, F ancesca Luzi 2, Alessand o Di Michele 3, S e ania Scuo a 4,
Sa a P ima illa 4, Ma ia Rachele Cecca ini 1, Tommaso Becca i 1, Césa An onio Vise as Ibo a 5,
Daniele Ramella 6, Mau izio Ricci 1and Luana Pe ioli 1,*


Ci a ion: Pagano, C.; Puglia, D.;
Luzi, F.; Michele, A.D.; Scuo a, S.;
P ima illa, S.; Cecca ini, M.R.; Becca i, T.;
Ibo a, C.A.V.; Ramella, D.; e al.
De elopmen and Cha ac e iza ion o
Xan han Gum and Algina e Based
Bioadhesi e Film o Pycnogenol
Topical Use in Wound T ea men .
Pha maceu ics 2021,13, 324.
h ps://doi.o g/10.3390/
pha maceu ics13030324
Academic Edi o : Ian S. Blagb ough
Recei ed: 27 Janua y 2021
Accep ed: 25 Feb ua y 2021
Published: 3 Ma ch 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/).
1Depa men o Pha maceu ical Sciences, Uni e si y o Pe ugia, 06123 Pe ugia, I aly;
[email p o ec ed] (M.R.C.); [email p o ec ed] (T.B.); [email p o ec ed] (M.R.)
2Ci il and En i onmen al Enginee ing Depa men , Uni e si y o Pe ugia, 05100 Te ni, I aly;
[email p o ec ed] (D.P.); [email p o ec ed] (F.L.)
3Depa men o Physics and Geology, Uni e si y o Pe ugia, 06123 Pe ugia, I aly;
[email p o ec ed]
4Is i u o Zoop o ila ico dell’Umb ia e delle Ma che, 06126 Pe ugia, I aly; [email p o ec ed] (S.S.);
[email p o ec ed] (S.P.)
5Depa men o Pha macy and Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o G anada,
Campus o Ca uja, 18071 G anada, Spain; c ise as@ug .es
6Depa men o Chemis y, College o Science and Technology, Temple Uni e si y,
Philadelphia, PA 19122, USA; [email p o ec ed]
*Co espondence: [email p o ec ed] (C.P.); [email p o ec ed] (L.P.)
Abs ac :
Pycnogenol (PYC) is a concen a e o phenolic compounds de i ed om F ench ma i ime
pine; i s biological ac i i y as an ioxidan , an i-in lamma o y and an ibac e ial sugges s i s use in he
ea men o open wounds. A bioadhesi e ilm, loaded wi h PYC, was p epa ed by cas ing, s a -
ing wi h a combina ion o wo biopolyme acqueous solu ions: xan han gum (1% w /w ) and sodium
algina e (1.5% w /w ), in a 2.5/7.5 (w /w ) a io. In bo h solu ions, glyce ol (10% w /w ) was added
as plas icizing agen . The ilm esul ed in an adhesi e capable o abso b a simula ed wound luid
(~ 65% w /w wi hin 1 h), he e o e sui able o exuding wounds. The mechanical cha ac e iza ion
showed ha he ilm is de o mable (elas ic modulus E = 3.070
±
0.044 MPa), sugges ing adap abili y
o any ype o su ace and esis ance o mechanical solici a ions. PYC is eleased wi hin 24 h by a sus-
ained mechanism, achie ing a maximum concen a ion o ~0.2 mg/mL, ha is sa e o ke a inocy es,
as shown by cy o oxici y s udies. A concen a ion o 0.015 mg/mL is eached in he i s 5 min a e
applica ion, a which poin PYC s imula es ke a inocy e g ow h. These p elimina y esul s sugges
he use o PYC in o mula ions designed o opical use.
Keywo ds: pycnogenol; xan han gum; sodium algina e; hyd ogel ilm; bioadhesion; wounds
1. In oduc ion
Pycnogenol (PYC) is he egis e ed ade name o a special s anda dized ex ac
ob ained om he ba k o he F ench ma i ime pine, Pinus pinas e ssp., species A lan ica,
amily Pinaceae, genus Pinus. I is g own in la ge monocul u es, especially in he Sou h-
wes e n F ench a ea o Biscay [1].
PYC is a concen a e o phenolic compounds (phenolic acids, ca echin, epica echin,
axi olin and p ocyanidins), p esen in bo h he ee and he glycosyla ed o ms [1].
These molecules a e esponsible o PYC’s biological ac i i y, as i has been known
since he ancien e a. I was men ioned by Hippoc a es as a emedy o in lamma o y
diseases, and in he Thesau us Medicaminum (1479) as a wound healing adju an [2].
The pha macological ac i i y o PYC has been epo ed in se e al s udies, du -
ing which bo h adical-sca enging and an i-in lamma o y p ope ies we e obse ed [
1
,
3
–
7
].
Pha maceu ics 2021,13, 324. h ps://doi.o g/10.3390/pha maceu ics13030324 h ps://www.mdpi.com/jou nal/pha maceu ics
Pha maceu ics 2021,13, 324 2 o 18
The la onoids which can p e en ee adicals om o ming esonance-s abilized phe-
noxyl adicals a e esponsible o PYC’s an ioxidan p ope ies [
8
]. The an i-in lamma o y
ac i i y can ins ead be asc ibed o PYC’s abili y o up- egula e he exp ession o gene
coding o 5-lipoxygenase and cyclooxygenase-2, as well as inhibi phospholipase A2 [9].
Mo eo e , PYC’s an ibac e ial ac i i y owa d g am-posi i e (as E. aecalis,Clos id-
ium pe ingens, S. au eus) and g am-nega i e (as E. coli,K. pneumoniae,P. ae uginosa) bac e ia
was obse ed [10].
Recen s udies highligh ed ha PYC can p omo e he syn hesis o molecules p esen
in he ex acellula ma ix such as hyalu onic acid and collagen [6].
The combina ion o he an ioxidan , an i-in lamma o y and an ibac e ial ac i i ies com-
bined wi h he s imula ion o ex acellula ma ix egene a ion, makes PYC an in e es ing
p oduc o use in wound ea men o mula ions.
Se e al s udies abou he p esence o PYC in opical p oduc s, such gels and c eams
in ended o wound applica ion a e p esen in li e a u e [
7
,
11
]. Howe e , such o mula ions
show a limi ed esidence ime and a e no able o p o ec he damaged a ea.
Fo his eason, he use o ad anced o mula ions is necessa y o pe o m a p omp
wound ea men , p e en ing bac e ia in asion o he damaged skin and se e e in lam-
ma ion ac o s esponsible o delayed healing. Wi h hese aspec s in mind, a sui able
o mula ion o wound ea men should: (i) con ain an ac i e ing edien able o p omo e
he epai p ocess, (ii) co e he wound p o ec ing i om mechanical damage and bac e ial
in asion, and (iii) emo e he excess exuda e. Recen s udies epo in-si u gel o ming
sys ems loaded wi h PYC [12] as iable al e na i es.
The pu pose o his s udy was o de elop an e ec i e biocompa ible o mula ion ha
would be sa e o he pa ien and en i onmen ally iendly. Thus, ilms we e ealized using
wo biopolyme s: xan han gum and sodium algina e, FDA app o ed as G.R.A.S. (gene -
ally ecognized as sa e) [
13
–
16
]. The s udy was di ided in h ee s eps: (i) iden i ica ion
o he bes ilm composi ion and p epa a ion me hod, (ii) in es iga ion o unloaded ilm
cha ac e is ics, and (iii) PYC loading and s udy o he loaded ilm pe o mances.
2. Ma e ials and Me hods
2.1. Ma e ials
Xan han gum was pu chased by Mul iagency S.n.c. (Ca a Mana a, PV, I aly). Al-
ginic acid sodium sal , calcium chlo ide dihyd a e we e supplied by Sigma Ald ich (Milano,
I aly). Pycnogenol (PYC) d y ex ac i . F ench ma i ime pine 65% OPCS was supplied by
A.C.E.F. s.p.a, Fio enzuola d’A da (Piacenza, I aly). Magnesium chlo ide was pu chased
om Ca lo E ba Reagen s S. .l. (Milano, I aly). Ul apu e wa e was ob ained by e e se
osmosis p ocess in a MilliQ sys em Millipo e (Roma, I aly). O he eagen s and sol en s
we e o analy ical g ade and used wi hou u he pu i ica ion. The pH 6.5 simula ed
wound luid (SWF) was p epa ed by dissol ing 8.30 g o NaCl and 0.28 g o CaCl
2
in
1000 mL o ul apu e wa e [17].
2.2. Me hods
2.2.1. Film P epa a ion
Films we e p epa ed by sol en cas ing me hod [
17
] s a ing om bina y mix u es
o biopolyme -based hyd ogels o alginic acid sodium sal (AL) and xan han gum (XG)
glyce ol (10% w ) used as plas icizing agen o he inal ilms. The AL based hyd ogel
was p epa ed unde magne ic s i ing (600 pm) by dispe sing he biopolyme in he wa e
p e iously added by glyce ol. XG based hyd ogel was p epa ed using mo a and pes le.
XG was p e iously we ed wi h glyce ol and hen hyd a ed wi h bidis illed wa e .
As a as loaded ilms a e conce ned, AL and XG, 5% w /w o PYC, hyd ogels we e
solubilized in he bidis illed wa e la e used o hyd ogel p epa a ion [11].
Film p o o ypes we e ob ained using bina y mix u es o (w /w ) o AL/XG hyd ogels
in di e en a ios. To emo e he ai inco po a ed du ing he mixing, AL and XG hyd ogel
and he co esponding blends we e degassed by an ARE-250 mixe (THINKY, Kidling on,
Pha maceu ics 2021,13, 324 3 o 18
England) a 2000 pm o 3 min (mixing) and a 2000 pm o 5 min (de oaming), a oom
empe a u e (RT). The hyd ogel mix u e (56.0 g) was cas ed in o ci cula Te lon moulds
(diame e 14 cm) and placed in he o en a 37.0
◦
C
±
0.1 o 24 h. A e wa ds, he ilms
we e ea ed wi h o a 5% (w / ) solu ion o CaCl
2·
2 H
2
O (6.0 mL) and placed again in he
o en a 37.0
◦
C
±
0.1 o u he 24 h. A e his ime, he d ied ilms we e emo ed om
he mould and s o ed unde CaCl2.
2.2.2. Film S o age Condi ions
Th ee s o age condi ions we e e alua ed o op imize he p ese a ion o he ilms’
o iginal p ope ies:
(1)
CaCl2( ela i e humidi y, R.H. 40%) a R.T.,
(2)
sa u a ed MgCl2solu ion a RT (R.H. 33%),
(3)
sa u a ed MgCl2solu ion a 4.0 ◦C (R.H. 34%).
2.2.3. The mog a ime ic Analyses
The mog a ime ic measu emen s o aw ma e ials and ilms we e pe o med by
using an Exs a 6300 TG/DTA sys em (Seiko, Woodland, CA, USA). Each ilm was cu in
simila po ions (weigh 10 mg) and placed inside small alumina c ucibles, unde con olled
and ine (ni ogen low, 200 mL/min) a mosphe e. The esidual mass o all he ilms a e
1 week o s o age in CaCl2desicca o s was measu ed a bo h 100 ◦C and 600 ◦C.
2.2.4. Mechanic Cha ac e iza ion
The ensile es s we e pe o med by a digi al mic op ocesso ins umen LLlyod
LR30K (Hampshi e, USA) The ilms we e cu in po ions o 100 mm
×
10 mm (UNI ISO
527) o p epa e samples wi h a use ul leng h o 50 mm. The expe imen s we e pe o med
a 5 mm/min, cell load 50 N. Values o maximum s ess, de o ma ion a b eak and elas ic
modulus we e egis e ed. The epo ed esul s a e an a e age o i e measu emen s (n = 5).
The samples we e placed in desicca o con aining a sa u a ed MgCl
2
solu ion o 1 week a
RT un il eaching cons an weigh .
2.2.5. Mo phology and Thickness
Film mo phology and hickness we e e alua ed by FE-SEM LEO 1525 ZEISS (Ca l Zeiss
Mic oscopy, Jena, Ge many). The samples we e p epa ed by deposi ion o he sample on
conduc i e ca bon adhesi e ape and hen me alized wi h ch omium (8 nm) by spu e ing.
2.2.6. Wa e Con en
To measu e he wa e con en , each ilm was cu in squa es o 4 cm
2
and d ied, and he
weigh loss was calcula ed. Each po ion was placed in h ee di e en condi ions:
(1)
en ila ed o en a 42 ◦C,
(2)
desicca o unde CaCl2,
(3)
desicca o unde P2O5.
Each sample was weigh ed be o e he expe imen (Wi) and a se imes (W ) o s o age
in he abo e-desc ibed condi ions (n = 3,
±
SD). The weigh % was calcula ed by using
Equa ion (1):
Weigh % =
Wi −W
Wi ×100 (1)
whe e Wiis he ini ial weigh o he ilm and W is he weigh a e s o age.
2.2.7. Wa e Holding S udies
Film abili y o abso b exuda es was e alua ed by means o hyd a ion pe cen age (%)
and ma ix e osion (DS) calcula ed by Equa ions (2) and (3), espec i ely:
Hyd a ion % =
W2 −W1
W2 ×100 (2)
Pha maceu ics 2021,13, 324 4 o 18
DS =
W1 −W3
W1 ×100 (3)
Each ilm was cu in po ions o 4 cm
2
(2 cm
×
2 cm) and a single po ion was weigh ed
(W1), imme sed in SWF (5 mL) inside a cen i uge ube (50 mL Co ning, To ino, I aly)
and held a 32.0
±
0.1
◦
C o es ablished imes (1, 2, 3, 4, 5, 6, 24, 48 h). A e imme sion,
he ilms we e wiped using il e pape o emo e he excess su ace SWF, and weigh ed
(W2). A e hyd a ion, he ilms we e d ied a 60
◦
C o 24 h, main ained o e CaCl
2
(RH 40%) o 48 h and eweigh ed (W3) [17].
2.2.8. Ex Vi o Adhesion S udies
Film abili y o bind he skin was e alua ed ex i o using samples (shoulde egion)
ob ained om pigs (La ge Whi e, weigh
∼
165–175 kg, u nished by Ve e ina y Se ice
o ASL N. 1 Ci àdi Cas ello, Pe ugia, I aly). The skin samples we e used o he assays
wi hin 12 h om pig dea h [
18
]. The ilm was a ached on a suppo using cyanoac yla e
glue and connec ed o he dynamome e Dida onic (Wha man GmbH, Dassel, Ge many).
A piece o po cine skin issue was ixed wi h cyanoac yla e glue on he su ace o a glass
suppo placed in a he mos a ic ba h a 32.0
±
0.5
◦
C. E e y ilm was cu in po ions o
2 cm
×
2 cm. The ee side o he skin was we ed wi h 50
µ
L o SWF and pu in con ac
wi h he ilm sample by applying a ligh o ce o 1 min. The o ce and ime necessa y o
de achmen o he ilm om he skin was measu ed and exp essed as he a e age o h ee
measu emen s (n = 3).
2.2.9. Release S udies
The dissolu ion es s o ansde mal pa ches using he ex ac ion cell (dep h o
2.6 mm, diame e 27 mm, elease su ace exposed 3.14 cm
2
) p esc ibed by he Eu opean
Pha macopoeia (Ph. Eu . 10
h
Ed.) was used o e alua e PYC elease om he ilm. The es
was pe o med o 24 h wo king a 40 pm in sink condi ions using SWF as dissolu ion
medium (400 mL) kep a 32.0
±
0.5
◦
C. A p ese in e als, samples (2 mL) we e ex ac ed,
eplaced by an equal amoun o SWF and analyzed by UV–Vis spec ophome e Agilen
8453 (Agilen Technologies, Ge many) using a calib a ion cu e in SWF (
λmax
= 281.0 nm;
2= 0.99) [17].
2.2.10. An imic obial Ac i i y
The an imic obial ac i i y o PYC solu ion and PYC loaded ilms was e alua ed by
a p ope ly adap ed aga di usion me hod [
17
]. The assay was pe o med on he s ains
epo ed in Table S1.
Each s ain (lyophilized) was suspended in 1.0 mL o s e ile demine alized wa e and
hen sown in sheep’s blood aga (CM 0271: p o eose pep one 15.0 g/L, li e diges 2.5 g/L,
yeas ex ac 5.0 g/L, sodium chlo ide 5.0 g/L, aga 12.0 g/L, s e ile sheep blood 50.0 mL/L,
pH 7.4
±
0.2 a 25.0
◦
C, OXOID, The mo Fishe , Fe en ino, I aly) o ob ain isola ed colonies,
ha we e a e wa ds incuba ed in condi ions speci ic o each s ain (Table S1). A e ha , a
b o h cul u e in BHI (bee hea in usion solids 17.5 g/L, p o eose pep one 10.0 g/L, glucose
2.0 g/L, sodium chlo ide 5.0 g/L di-sodium phospha e 2.5 g/L, pH 7.4
±
0.2 a 25.0
◦
C) was
p epa ed om he colony o each s ain and incuba ed o e nigh a
37.0 ±0.1 ◦C
. The mi-
c oo ganisms we e hen coun ed o de e mine he op imal dilu ion o he expe imen .
The cul u e medium employed o e alua ion o he an ibac e ial ac i i y (mea ex ac
3.0 g/L, mea pep one 5.0 g/L, glucose 4.0 g/L, sodium chlo ide 10.0 g/L, di-po assium
phospha e 1.0 g/L, aga noble 13.0 g/L, pH 7.2
±
0.2 a 25
◦
C) was dissol ed a 100
◦
C,
cooled o 44–47
◦
C and insemina ed wi h 1.0 mL o bac e ial suspension o ob ain a inal
concen a ion o 10
5
cells/mL. This suspension was accu a ely mixed and pou ed (25 mL)
in o Pe i dishes (90 mm diame e ), le i cool on a ho izon al su ace. A he ime o use,
PYC was dilu ed wi h s e ile demine alized wa e o ob ain i e concen a ions: 10, 1, 0.1,
0.05 and 0.025 mg/mL.
Pha maceu ics 2021,13, 324 5 o 18
Fo loaded ilms, he expe imen was pe o med as ollows: a small squa e (
1 cm ×1 cm
)
o he wo ilms was placed in each se ies o pla es, simila o how he ac i e ing edien ee
co esponding ilms we e es ed. Th ee aga pla es, uninocula ed, we e incuba ed o e i y
medium s e ili y. The pla es incuba ed in he condi ions epo ed in Table S1, we e hen
measu ed o inhibi ion halos by a gauge [17].
2.2.11. Cy o oxici y Assay
The HaCaT cell line (300493, CLS Cell Lines Se ice, pu chased om I.Z.S.l.E.R.
(Is i u o Zoop o ila ico Spe imen ale della Lomba dia e dell’Emilia Romagna, I aly)) was
used as a ep esen a i e model o app ecia e he epide mal homeos asis and healing du ing
wound ea men . HaCaT, a monolaye human immo alized ke a inocy e, was pu chased
om I.Z.S.I.E.R a 46
◦
passage le el. The cellula iabili y was assessed using MTT assay
a e 24 h o ea men [
19
]. HaCaT cells was used always be ween 48
◦
–55
◦
passage and
each expe imen was pe o med in iplica e o wo imes. Fo MTT assay a 96-well pla e
was seeded. The inal cell densi y was 1
×
10
4
cells/well. A e 24 h, when he cells
eached he 60% o con luence, esh comple e medium was eplaced o ea men wi h
PYC samples dilu ions om he s ock solu ion p epa ed incuba ing he ilm (1
×
1 cm)
wi h DMEM (10 mL) o 24 h.
MTT eagen (0.5 mg/mL in PBS) was added in each well a 0.05
µ
g/
µ
l inal con-
cen a ion o 3 h. Then, he supe na an was ca e ully emo ed, and he OD alues
we e measu ed spec opho ome ically (Eliza MAT 2000, DRG Ins umen s GmbH, Ma -
bu g, Ge many) and cell iabili y was exp essed as a pe cen age ela i e, as p e iously
desc ibed [20].
2.2.12. In i o Wound Healing Assay
Cy oSelec
™
Wound Healing Assay Ki (Cell Biolabs, Inc., San Diego, CA, USA) was
pu chased o in es iga e he e ec o PYC eleased om he ilm on wound closu e
in i o
.
A 24-weels issue cul u e pla e con aining p ope ly ea ed inse s was used.
HaCaT cells o hese expe imen s was seeded in DMEM comple e medium a he
inal concen a ion o 5
×
10
4
/500
µ
L (1
×
10
5
/mL). A e 24 h, when ke a inocy e eached
80% o con luence, he inse s we e emo ed om he wells lea ing he wound ield [21].
The cells we e ea ed wi h he wo di e en concen a ions o PYC ob ained incuba -
ing he ilm (1
×
1 cm) wi h DMEM (10 mL) o 24 h (0.015 mg/mL and 0.030 mg/mL),
o 24 u he hou s [22].
Mig a ion in o he wound ield was de e mined as p e iously desc ibed and pic u es
o con ol cells (CTR) and ea ed cells (PYC) a e 6, 12 and 24 h we e aken and h ee
ields o each condi ion we e compa ed [22].
The o al wound ield su ace a ea was calcula ed conside ing he dimensions o he
inse : To al Su ace A ea = 0.9 mm (leng h)
×
1.8 mm = 1.62 mm
2
. To measu e he %
closu e, he mig a ion cell su ace a ea was de e mined o each expe imen (Mig a ion Cell
Su ace = leng h o cell mig a ion ×2×1.8 mm).
The pe cen closu e o wound ield was calcula ed conside ing h ee di e en imes o
ea men : 6, 12 and 24 h and using Equa ion (4):
% closu e =
mig a ion cell su ace
o al su ace a ea ×100 (4)
2.2.13. S a is ical Analysis
Resul s we e epo ed as mean
±
s anda d de ia ion (mean
±
SD). One-way ANOVA
es was used o s a is ical analysis. Di e ences we e conside ed s a is ically signi ican
o p< 0.05.
3. Resul s and Discussions
Bioadhesi e ilms a e use ul o o e come p oblems commonly epo ed abou con en-
ional wound d essings. One o he main limi a ions is he use o adhesi es o p omo e he

Pha maceu ics 2021,13, 324 6 o 18
adhesion o skin esul ing in a pain ul and auma ic emo al wi h consequen damage o
he su ounding issue. The de elopmen o bioadhesi e medica ions, based on biopoly-
me s and hus easily emo able by washing, could be a sui able al e na i e. Wi h his goal
in mind, he ocus was shi ed o he use o na u al biopolyme s, app o ed by FDA and
EMA and used in p oduc s o heal h ca e and hus sa e o use (classi ied as G.R.A.S.).
Films based on he use o biopolyme s could allow a sui able wound ea men due o
p ope ies such as compa ibili y wi h issues, high abili y o hold wa e , and o p o ide
a mois en i onmen p o ec ing he wound om desicca ion, in ec ions and mechanical
solici a ions [23].
The biopolyme s o he de elopmen o he bioadhesi e ilms we e selec ed acco ding
o he ollowing se equi emen s: (i) adhesion capaci y o skin su ace [
24
], (ii) high
esidence ime in he applica ion si e, (iii) easy and a auma ic emo al (e.g., by washing),
(i ) sus ained elease o he ac i e ing edien , ( ) mechanical p o ec ion o he damaged
a ea [
25
], and ( i) eco- iendly. Xan han gum (XG) was ound o be a iable ma e ial;
i displays good wa e -solubili y and excellen biocompa ibili y, and i is non- oxic and no
i i an o he skin. I was he e o e chosen as he op imal polyme o ilm p epa a ion.
Ini ially, hyd ogels based on XG (0.5%, 1% and 2% w /w ) we e p epa ed and used in
ilm o ma ion. Howe e , unsa is ying esul s we e ob ained and i s mixing wi h ano he
na u al biopolyme was de ised. Sodium algina e (AL) was selec ed because o i s use as
gelling, hickening and ilm o ming agen [26].
3.1. Unloaded Film P epa a ion and Cha ac e iza ion
A e p elimina y s udies based on he e alua ion o hyd ogels cha ac e is ics (homo-
genei y and consis ency), easy o cas ing and inal ilm appea ance (impe ec ion de ec ed
by isual inspec ion), he mos sui able composi ions o he s a ing hyd ogels used o
p epa e he ilms we e he ollows: hyd ogel-AL: AL 1.5% (w /w ), glyce ol 10% (w /w ),
bidis illed wa e un il 100 g; hyd ogel-XG: XG 1% (w /w ), glyce ol 10% (w /w ), bidis-
illed wa e un il 100 g. Glyce ol was chosen as plas icizing agen as obse ed in o he
s udies [17,27].
Di e en a ios o hyd ogel-AL/hyd ogel-XG (5.0/5.0; 1.5/8.5; 8.5/1.5; 7.5/2.5; 2.5/7.5;
2.0/8.0; 1.0/9.0 w /w ) we e conside ed and a p elimina y e alua ion based on bo h easy
p oduc ion (bubbles emo al and cas ing) and ilm inal p ope ies (adhesion o skin, lexi-
bili y, esis ance o ac ion, applica ion, and emo al by washing) was pe o med. In he
end, he hyd ogels showing he bes composi ions we e A and B (Table 1); he co espond-
ing ilms (Film A and Film B) we e hus p oduced and ully cha ac e ized.
Table 1. Composi ions o he hyd ogel mix u es (AL/XG) chosen.
Hyd ogel AL
(% w /w )
XG
(% w /w )
Glyce ol
(% w /w )
Bidis illed Wa e
(% w /w )
A 2.50 7.50 10.00 80.00
B 1.50 8.50 10.00 88.00
3.2. S o age Condi ions
S o age condi ions ep esen a c i ical poin o ilms; modi ica ions o wa e con en
du ing he shel li e could dec ease i pe o mance du ing he applica ion phase. Inad-
equa e empe a u e and humidi y condi ions could be esponsible o so ening and/o
s i ening. Wi h his in mind, he p epa ed ilms we e emo ed om he mould and placed
in desicca o s unde a di e se ange o s o age condi ions (Table S2) and submi ed o isual
inspec ion and wa e con en de e mina ion a e 7 days. All he indica ions de i ing om
his assay can he e o e be use ul o planning he sui able packaging o he o mula ion.
S o age in a close sys em (desicca o ) was in es iga ed using CaCl
2
and MgCl
2
as
hey a e he mos commonly used desiccan s [
28
,
29
]. Films s o ed unde sa u a ed MgCl
2
solu ion a 4
◦
C appea ed gela inous and he e o e di icul o handle and s icky in com-
pa ison o a esh ilm; his was p obably due o ehyd a ion and gela ion- ype e ec s on
Pha maceu ics 2021,13, 324 7 o 18
algina e. The s o age condi ions unde CaCl
2
and unde sa u a ed MgCl
2
solu ion a RT
esul ed as he mos sui able ones, wi hou signi ican modi ica ions o he ilms du ing
s o age. S o age unde CaCl
2
was chosen o u he s udies based on he e alua ion o
wa e con en in he ilms.
3.3. Wa e Con en Measu emen
The e alua ion o he esidual wa e con en in he ilms is impo an , as i in luences
he pe o mances o he o mula ion in e ms o lexibili y, adhesi i y and mechanical
p ope ies. The amoun o wa e in ilms A and B a e s o age unde CaCl
2
was measu ed
by wo di e en app oaches. The i s app oach consis ed in he measu emen o he wa e
con en by dynamic he mog a ime ic cu es (TGA), while he second es ima ion was
made by means o weigh loss calcula ions a e iso he mal s o age a di e en cons an
empe a u es in he o en o 24 h. Resul s o he mog a ime ic analysis (TGA) o XG,
AL and glyce ol as aw ma e ials a e epo ed in Figu e S1, while TG and DTG he mo-
g ams o he ilms a e included in Figu e 1A,B. These expe imen s we e pe o med o
calcula e he ola ile con en o he di e en ma e ials a low empe a u es; speci ically,
he measu emen s we e done o es ima e he esidual wa e con en and ela e i o ilm
p ope ies [30].
Figu e 1. TGA and DTA p o iles o ilm A and ilm B (n= 3).
AL he mog am displayed wo dis inc s ages (Figu e S1a). The i s one, in he
ange o 30–160
◦
C, wi h a maximum decomposi ion a e a 104.0
◦
C, is a ibu able o
elimina ion o wa e adso bed by he hyd ophilic polyme . The second one, in he ange o
210–310
◦
C wi h a maximum decomposi ion a e a 255.7
◦
C (Figu e S2b), was asc ibed
o a complex p ocess including dehyd a ion o he saccha ide ings, depolyme iza ion
wi h he o ma ion o wa e , CO
2
and CH
4
. The empe a u e, a which 50% weigh loss
happens, was ound o be 300
◦
C o AL [
30
]. XG he mog am showed single s ep he mal
deg ada ion, ollowing an ini ial weigh loss due o he emo al o mois u e. The polyme ic
he mal deg ada ion s a s a 200
◦
C; he main peak is cen ed a 302.0
◦
C wi h a weigh
loss o a ound 52% (Figu e S1a). The a e o weigh loss inc eases ini ially, bu a e 50%
weigh loss, he a e was ound o dec ease [
31
]. The he mog a ime ic analysis o glyce ol
showed single s ep he mal deg ada ion cen ed a 255.7 ◦C (Figu e S1a) [31].
In bo h cases, he esidual wa e con en was es ima ed o be below 4% a 100.0
◦
C
(Figu e 1A, esidual mass cu e). Figu e 1B shows he de i a i e cu es (DTG) o nea ilm
A and B, cha ac e ized by he p esence o a mul i-s ep deg ada ion beha iou . The i s
peak, cen ed a 100.0
◦
C, is a ibu ed o he e apo a ion o wa e con en . Assumed ha
he d ied XG and AL did no unde go weigh losses du ing hea ing [
32
], he measu ed
weigh loss below 100.0
◦
C can be exclusi ely asc ibed o wa e con en ; no subs an ial
di e ences we e ound o he wo di e en ly o mula ed ilms. A highe empe a u es,
he ilms show one main deg ada ion s ep a 240–280
◦
C, a ibu ed o he main ac ions
in he ilms composi ion i.e., glyce ol, XG and AL. The hi d peak a a ound 400.0
◦
C is
ela ed o he p esence o algina e componen [23].
Pha maceu ics 2021,13, 324 8 o 18
The ilm’s wa e con en was also calcula ed by measu ing he weigh modi ica ions
unde di e en s o age condi ions and applying Equa ion (1). Table 2shows ha s o age
in he o en a 42
◦
C allows he highes emo al o wa e a e 24 h. On he o he hand,
he esul s ob ained using CaCl
2
and P
2
O
5
a e compa able. In all cases, he o e all wa e
emo al was e y low, sugges ing ha s o age in o en a 42
◦
C is he mos e icien me hod
o emo e esidual wa e . The esidual wa e con en emains high when compa ed o he
esul s ob ained om TGA measu emen , pe o med in dynamic hea ing condi ions.
Table 2. Films wa e loss ob ained a e one day o s o age a di e en condi ions.
Film S o age Condi ions Wa e Loss (%)
A
en ila ed o en a 42 ◦C 4.96 ±1.58
desicca o unde CaCl26.51 ±1.58
desicca o unde P2O56.10 ±3.35
B
en ila ed o en a 42 ◦C 4.33 ±1.01
desicca o unde CaCl29.77 ±3.76
desicca o unde P2O58.30 ±1.25
3.4. Mechanical Cha ac e iza ion
The mechanical cha ac e iza ion o ilms is essen ial as hese o mula ions we e de-
signed o be applied on skin and o con o m o e e y ype o su ace. Fo his eason,
mo e in o ma ion abou i s elas ic esponse is necessa y. The mechanical p ope ies o
hyd ophilic polyme s and o edible ma ices a e s ongly in luenced by RH as he humidi y
ac s as plas icize [33].
Mo eo e , ilms mus be esis an o mechanical solici a ions o which hey a e sub-
jec ed (e.g., du ing emo al om packaging, applica ion and pe iod o esidence on he
skin su ace). Acco ding o his, e alua ion o ensile p ope ies was pe o med by using
a dynamome e .
Fo each o mula ion maximum s ess (
σmax
), de o ma ion a maximum s eng h
(
εa σmax
) and elas ic modulus (E) we e measu ed (Table 3). The analysis o s ess-s ain
cu es o unloaded ilms (Figu e 2) showed ha ilm A is mo e de o mable han ilm B,
as con i med by he highe alue o s ain a b eak, sugges ing ha he composi ion o
ilm A could be use ul o he ixed objec i e. We ound ha a highe quan i y o algina e
induces an imp o emen o ensile cha ac e is ics, con ibu ing a de ec able inc ease in
maximum s eng h, elas ic modulus, and de o ma ion a b eak [34].
Table 3.
S ess a b eak (
σmax
), de o ma ion a b eak (
εa σmax
) and elas ic modulus (E) measu ed o
ilm A and ilm B; * p< 0.001; ** p> 0.05; *** p< 0.001 ilm A s ilm B.
Film AL/XG
(Ra io w /w ) σmax (MPa) εa σmax (%) E (MPa)
A 2.5/7.5 0.303 ±0.077 * 23 ±4 ** 2.823 ±0.148 ***
B 1.5/8.5 0.120 ±0.010 22 ±4 1.278 ±0.169
When used wi hou he addi ion o any plas icizing agen AL and XG show alues o
de o ma ion lowe han 3%; [
33
,
35
] he alues eques ed o no mal skin a e be ween 61
and 70% [
36
,
37
]. This p oblem can be o e come using glyce ol i s chemical s uc u e is able
o e ain wa e molecules, and s o ing in con olled-humidi y en i onmen . The amoun
o plas icize in ilms A and B is, espec i ely, 8.9 and 9.3 imes g ea e han he o al
quan i y o he polyme ic ma ix. Such a high amoun o plas icize g ea ly in luences
he mechanical esponse o he o e all sys ems. As he AL con en inc eases, high alues
o ensile s ess a b eak a e measu ed (Table 3); his is in ag eemen wi h o he au ho s’
s udies [
33
], sugges ing ha AL is he main esponsible o ilm elas ici y and de o mabili y.
Pha maceu ics 2021,13, 324 9 o 18
Figu e 2. S ess- s ain cu ed o ilm A ( ed line) and ilm B (black line), (n= 5).
3.5. Mo phology and Thickness
The mo phology and he hickness o bo h ilms we e s udied by scanning elec on mi-
c oscopy (Sec ion 2.2.5). The wo ilms show simila mo phology ( ilm A:
Figu e 3A,B
and
ilm B: Figu e 3D,E); in pa icula , a w inkled su ace is de ec able. The su ace oughness
is an impo an p ope y o bioadhesi e sys ems as i inc eases he su ace a ea a ailable
o adhesion. The ilms’ hickness was also measu ed (Figu e 3C,F, espec i ely); ilm A
esul ed mo e compac and hinne (410
±
2.5
µ
m) compa ed o ilm B (
529.4 ±8.7 µm
).
This di e ence is p obably a ibu able o he high AL con en in ilm A, esponsible o a
mo e compac and e icula ed s uc u e.
Figu e 3. Mic og aphs o he su ace o ilm A (A,B) and ilm B (D,E); hickness o ilm A (C) and ilm B (F) (n= 3).
3.6. Wa e Holding S udies
The ilms’ abili y o abso b wound exuda e was e alua ed
in i o
by wa e holding
s udies. The ob ained esul s (Figu e 4A) show ha bo h ilms hyd a e apidly a e
con ac wi h SWF. A e 1 h, he amoun o abso bed SWF is ~ 65% w /w o bo h es ed
o mula ions. In he case o ilm A, his alue is main ained un il he end o he expe imen
(48 h), sugges ing ha he wa e up ake is immedia e and does no change. Fo ilm B
ins ead, i inc eases sligh ly o 72% a e 48 h.
Pha maceu ics 2021,13, 324 16 o 18
The bes esul is achie ed a e 24 h o ea men wi h he lowes PYC concen-
a ion es ed (0.015 mg/mL). In ac , he wound is comple ely closed (100% o clos-
ing). In e es ing di e ences be ween he wo assessed concen a ions, in compa ison o
con ol cells (CTR), a e ob ained a e 6 and 12 h o ea men . Compa ed o he CTR,
he abili y o s imula e cell g ow h can be obse ed a e bo h 6 and 12 h o ea men .
Wi hin 6 h
, PYC 0.015 mg/mL and PYC 0.03 mg/mL exhibi pe cen closu es equal o
62.5% ±2.6%
, and 57.9
±
1.8%, espec i ely; he i s one han he one measu ed o
he CTR (
54.7% ±5.1%
). I is in e es ing o no e ha a e 12 h, cells ea ed wi h he
0.015 mg/mL PYC solu ion show a dec eased a ea o he wound ield (88.9
±
3.2% o
closing). Ins ead, such enhanced healing ac i i y was no ound in he cells ea ed wi h he
highe PYC concen a ion (0.030 mg/mL), which achie es only 75.1%
±
4.4% (compa able
o CTR 72.1%
±
7.8%). A he 24-h end-poin , wound closu e nea s 100% o bo h PYC
concen a ions (Figu e 11) s CTR in which he wound ield is s ill open.
4. Conclusions
Picnogenol (PYC) is a iable molecule o wound ea men . I was o mula ed in
bioadhesi e ilms ob ained om a mix u e o he biopolyme s xan han gum and alginic
acid sodium sal hyd ogels. The ilm showed sui able mechanical p ope ies such as high
de o mabili y, sugges ing easy adap abili y o any ype o su ace. The ilm composi ion
was ound o be capable o easy adhesion o skin and o abso bing he exuda es om
he wound.
In i o
assays demons a ed ha he de eloped ilms a e ac i e agains he
S. pyogenes,S. au eus and E. aecalis bac e ial s ains. The sus ained elease o PYC om
he o mula ion sugges s ha his o mula ion could be applied once-pe -day, allowing a
comple e p o ec ion o he damaged a ea and p omo ing he healing also by s imula ing
ke a inocy es g ow h.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h ps://www.mdpi.com/1999-4
923/13/3/324/s1, Figu e S1: i le, Table S1: i le, Video S1: i le.G ow h condi ions o he s ains
used o he an imic obial ac i i y assay; Table S2. Di e en condi ions assayed o ilm s o age
and obse a ions a e 7 days; Figu e S1. TGA p o iles o AL, XG and glyce ol (a); DTG p o iles
o AL, XG and glyce ol (b); Figu e S2. (a) S. au eus; (b) S. epide midis; (c) E. aecalis; (d) B. sub ilis;
(e) S. pyogenes
; Figu e S3. Film A-loaded: (a) E. aecalis, (b) S. pyogenes, (c) S. au eus; Figu e S4.
Viabili y measu ed
in i o
on HaCaT cells incuba ed wi h di e en olumes o DMEM p e iously
incuba ed o 24 h wi h he pa ch (2
×
2 cm in 10 mL o DMEM) ee om PYC. CTR, un ea ed cells
in DMEM we e se a 100%. DMSO in h ee di e en pe cen ages (1%, 2% and 4%) as posi i e
con ols (n= 3).
Au ho Con ibu ions:
Me hodology, C.P., A.D.M., S.P., S.S., F.L., M.R.C.; alida ion, C.P., A.D.M.,
D.P., T.B., S.P.; o mal analysis, C.P., A.D.M., F.L., S.P., M.R.C.; in es iga ion, L.P., M.R., D.P., S.P.,
S.S., A.D.M.; esou ces, L.P., D.P., A.D.M., S.S; da a cu a ion, A.D.M., F.L., D.P., C.P., S.P., C.A.V.I.;
w i ing—o iginal d a p epa a ion, C.P., L.P., F.L., C.A.V.I., T.B., D.R.; w i ing— e iew and edi ing,
C.P., L.P., M.R., D.P., F.L., M.R.C., D.R.; isualiza ion, C.P., L.P., D.P., L.P., M.R., T.B.; supe ision,
C.P., L.P., D.P., F.L., D.R.; p ojec adminis a ion, unding acquisi ion, L.P. All au ho s ha e ead and
ag eed o he published e sion o he manusc ip .
Funding: This esea ch ecei ed no ex e nal unding.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : No applicable.
Acknowledgmen s:
Au ho s since ely acknowledge Ma co Ma ani om he Depa men o Pha ma-
ceu ical Sciences o echnical assis ance, and Simone a De Angelis om ASL N. 1 (Ci àdi Cas ello,
Pe ugia, I aly) o p o iding pig skin samples.
Con lic s o In e es : The au ho s decla e no con lic o in e es .

Pha maceu ics 2021,13, 324 17 o 18
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