Imprinted Contact Lenses for Ocular Administration of Antiviral Drugs

polyme s
A icle
Imp in ed Con ac Lenses o Ocula Adminis a ion
o An i i al D ugs
Angela Va ela-Ga cia, JoséLuis Gomez-Amoza , Angel Conchei o and
Ca men Al a ez-Lo enzo *
Depa amen o de Fa macolog
í
a, Fa macia y Tecnolog
í
a Fa mac
é
u ica, I+D Fa ma G oup, Facul ad de Fa macia
and Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS), Uni e sidade de San iago de Compos ela,
15782 San iago de Compos ela, Spain; angela. a ela.ga [email p o ec ed] (A.V.-G.);
[email p o ec ed] (J.L.G.-A.); [email p o ec ed] (A.C.)
*Co espondence: ca men.al a ez.lo [email p o ec ed]; Tel.: +34-881815239
Recei ed: 7 Augus 2020; Accep ed: 2 Sep embe 2020; Published: 4 Sep embe 2020


Abs ac :
A a ie y o ocula diseases a e caused by i uses, and mos ea men s ely on he use
o sys emic o mula ions and eye d ops. The e icien ocula ba ie s ha oppose an i i al d ug
pene a ion ha e p omp ed he de elopmen o imp o ed opical deli e y pla o ms. The aim was
o design hyd ogel con ac lenses endowed wi h an a ini y o acyclo i (ACV) and i s p od ug
alacyclo i (VACV), i s -choice d ugs agains he pes simplex i us (HSV) ocula ke a i is, and ha
can sus ain he elease o he apeu ic doses du ing daily wea ing. Func ional monome s sui able
o in e ac ion wi h hese d ugs we e sc eened using compu a ional modeling. Imp in ed and
non-imp in ed hyd ogels we e p epa ed wi h a ious con en s in he unc ional monome me hac ylic
acid (MAA) and cha ac e ized in e ms o swelling, ansmi ance, mechanical p ope ies, and
ocula compa ibili y (hen’s egg es on cho ioallan oic memb ane (HET-CAM) assay). The alues
we e in he ange ypical o so con ac lenses. Compa ed o ACV, he capabili y o load VACV
was ema kably highe due o s onge elec os a ic in e ac ions wi h MAA. The ad an ages o
he imp in ing echnology we e e idenced o VACV. S abili y o VACV loading solu ion/hyd ogels
unde s eam hea s e iliza ion and subsequen d ug elease was in es iga ed. Pe meabili y s udies
h ough bo ine and po cine co nea and scle a o he d ug eleased om he hyd ogels e ealed ha
VACV accumula es in he co nea and can easily c oss he scle a, which may acili a e he ea men o
bo h an e io and pos e io eye segmen s diseases.
Keywo ds:
d ug-elu ing con ac lens; molecula ly imp in ed hyd ogel; an i i al d ug; sus ained
elease; co nea pene a ion; scle a pene a ion
1. In oduc ion
In ec ion by he pes simplex i us (HSV) s a s when he i us comes in o con ac wi h damaged
skin o mucous memb anes; he incuba ion pe iod ex ends o 4 days [
1
]. The e a e wo sub ypes
o he pes simplex i us, HSV-1 and HSV-2. The main di e ence is ha HSV-1 appea s mainly in
he o olabial a ea, while HSV-2 a ec s he geni al a ea, al hough, in de eloped coun ies, cases o
geni al condi ions due o HSV-1 and o olabial due o HSV-2 a e on he inc ease [
2
]. I is es ima ed ha
90% o he wo ld’s popula ion is in ec ed wi h HSV [
2
]. Pe iodically, he i us can eac i a e and a el
o he skin o mucous memb anes, causing a ecu en symp oma ic o asymp oma ic in ec ion. Many
ac o s can igge his eac i a ion, o example, s ess, exposu e o hea o cold, mens ua ion, e e ,
o immunosupp ession [
3
]. The clinical mani es a ions depend on whe he he in ec ion is p ima y o
ecu en , he immune s a us o he hos , and he en y po al [4].
Polyme s 2020,12, 2026; doi:10.3390/polym12092026 www.mdpi.com/jou nal/polyme s
Polyme s 2020,12, 2026 2 o 19
A he ocula le el, ecu en HSV ep esen s a se ious epidemiological cause o in ec ious
and in lamma o y disease [
5
,
6
]. He pes disease a ec s mo e han 10 million people, and o hese,
app oxima ely 2 million su e ision p oblems in he a ec ed eye. Epi helial ke a i is accoun s o
50–80% o ocula he pes. Wo ldwide, abou 1 million new o ecu en cases o epi helial ke a i is occu
annually [
7
], and i is he mos common cause o i e e sible blindness in de eloped coun ies [
8
].
Ocula he pes is ela ed o p ima y o o acial he pes (HSV-1); abou 56%–58% o pa ien s wi h ocula
he pes ha e a his o y o o al he pes [8,9].
Cu en he apy o he ea men o HSV ocula ke a i is includes opical, o al, and in a enous
an i i al agen s [
3
,
10
]. Vi al ke a i is can become a ch onic and ecu en disease, a ec ing pa ien s’
quali y o li e due o he limi ed e icacy o a ailable ea men s [
10
,
11
]. Mos o he app o ed an i i als
a e acyclic nucleosides and nucleo ide analogs, which in e up i us eplica ion [12].
Acyclo i (9-(2-hyd oxye hoxyme hyl) guanine) (ACV) is a pu ine nucleoside analog ha emains
he ea men o choice o HSV-1 in ec ions o da e [
10
,
11
]. I is a selec i e an i i al agen as i
speci ically a ge s i us-in ec ed cells and selec i ely inhibi s he i al DNA polyme ase [
4
,
13
].
Ne e heless, he inhibi ion o i us eplica ion may no a ec he la ency, so he in ec ion may
ha e no been sol ed [
3
,
10
,
12
]. ACV has a good sa e y p o ile and is well- ole a ed by pa ien s, bu
i s o al bioa ailabili y is low (10%–20%) and i s plasma hal -li e is sho , which in ol es equen
adminis a ions [
3
–
5
]. Mo eo e , he e a e s udies ha demons a e he g owing esis ance o his d ug
mainly in immunosupp essed subjec s, de eloped h ough a mu a ion o he i al gene hymidine
kinase, essen ial o he phospho yla ion o ACV. An addi ional limi a ion o o al adminis a ion
o ACV is enal oxici y in elde ly pa ien s, who a e unable o exc e e he d ug p ope ly [
8
,
10
,
12
].
An al e na i e is he opical applica ion o ACV, bu i s e ec i eness depends on i s abili y o c oss
he epi helium [
3
]. In compa a i e s udies wi h o he non-selec i e an i i al agen s, ACV oin men
has been shown o be mo e e ec i e and less oxic [
13
]. The p oblem wi h opical o ms is hei low
e en ion ime on he eye su ace [
10
]. In some cases, co icos e oids a e used as adju an he apy o
an i i als [
11
], bu many side e ec s can occu in long- e m he apy, including ca a ac , supp ession o
he immune esponse, and possible seconda y glaucoma [10,14].
Valacyclo i (VACV) is a l- aline es e o ACV (Figu e 1) and ac s as a p od ug wi h imp o ed
bioa ailabili y [
13
,
15
,
16
], bu he o al adminis a ion o VACV s ill does no p o ide e ec i e
concen a ions in he eye [
17
]. Fo example, o al adminis a ion o VACV (500 mg/day) does no
supp ess HSV-1 DNA shedding in ea s [
16
,
18
]. Repo s on opical o mula ions o VACV a e s ill sca ce
and, so a , hey ocused on ca ionic Eud agi mic osphe es o mucoadhesion o he co nea su ace [
15
]
and solid lipid nanopa icles o pene a e in o he eye issues [
19
]. Recen s udies ha e con i med
ha VACV binds o he oligopep ide anspo e o he co neal epi helium, and ha i s ansco neal
pe meabili y is h ee imes highe han ha o ACV. I is ans o med o ACV by enzyma ic hyd olysis
in he eye [
15
,
20
]. VACV also shows a highe a ini y han ACV o he amino acid anspo e ATB
0,+
p esen in ocula issues [21].
Polyme s 2020, 12, x FOR PEER REVIEW 2 o 19
50–80% o ocula he pes. Wo ldwide, abou 1 million new o ecu en cases o epi helial ke a i is
occu annually [7], and i is he mos common cause o i e e sible blindness in de eloped coun ies
[8]. Ocula he pes is ela ed o p ima y o o acial he pes (HSV-1); abou 56%–58% o pa ien s wi h
ocula he pes ha e a his o y o o al he pes [8,9].
Cu en he apy o he ea men o HSV ocula ke a i is includes opical, o al, and in a enous
an i i al agen s [3,10]. Vi al ke a i is can become a ch onic and ecu en disease, a ec ing pa ien s'
quali y o li e due o he limi ed e icacy o a ailable ea men s [10,11]. Mos o he app o ed
an i i als a e acyclic nucleosides and nucleo ide analogs, which in e up i us eplica ion [12].
Acyclo i (9-(2-hyd oxye hoxyme hyl) guanine) (ACV) is a pu ine nucleoside analog ha
emains he ea men o choice o HSV-1 in ec ions o da e [10,11]. I is a selec i e an i i al agen as
i speci ically a ge s i us-in ec ed cells and selec i ely inhibi s he i al DNA polyme ase [4,13].
Ne e heless, he inhibi ion o i us eplica ion may no a ec he la ency, so he in ec ion may ha e
no been sol ed [3,10,12]. ACV has a good sa e y p o ile and is well- ole a ed by pa ien s, bu i s o al
bioa ailabili y is low (10%–20%) and i s plasma hal -li e is sho , which in ol es equen
adminis a ions [3–5]. Mo eo e , he e a e s udies ha demons a e he g owing esis ance o his
d ug mainly in immunosupp essed subjec s, de eloped h ough a mu a ion o he i al gene
hymidine kinase, essen ial o he phospho yla ion o ACV. An addi ional limi a ion o o al
adminis a ion o ACV is enal oxici y in elde ly pa ien s, who a e unable o exc e e he d ug
p ope ly [8,10,12]. An al e na i e is he opical applica ion o ACV, bu i s e ec i eness depends on
i s abili y o c oss he epi helium [3]. In compa a i e s udies wi h o he non-selec i e an i i al
agen s, ACV oin men has been shown o be mo e e ec i e and less oxic [13]. The p oblem wi h
opical o ms is hei low e en ion ime on he eye su ace [10]. In some cases, co icos e oids a e
used as adju an he apy o an i i als [11], bu many side e ec s can occu in long- e m he apy,
including ca a ac , supp ession o he immune esponse, and possible seconda y glaucoma [10,14].
Valacyclo i (VACV) is a L- aline es e o ACV (Figu e 1) and ac s as a p od ug wi h imp o ed
bioa ailabili y [13,15,16], bu he o al adminis a ion o VACV s ill does no p o ide e ec i e
concen a ions in he eye [17]. Fo example, o al adminis a ion o VACV (500 mg/day) does no
supp ess HSV-1 DNA shedding in ea s [16,18]. Repo s on opical o mula ions o VACV a e s ill
sca ce and, so a , hey ocused on ca ionic Eud agi mic osphe es o mucoadhesion o he co nea
su ace [15] and solid lipid nanopa icles o pene a e in o he eye issues [19]. Recen s udies ha e
con i med ha VACV binds o he oligopep ide anspo e o he co neal epi helium, and ha i s
ansco neal pe meabili y is h ee imes highe han ha o ACV. I is ans o med o ACV by
enzyma ic hyd olysis in he eye [15,20]. VACV also shows a highe a ini y han ACV o he amino
acid anspo e ATB0,+ p esen in ocula issues [21].
Figu e 1. S uc u e o acyclo i (ACV) and alacyclo i (VACV) and wo k low o he expe imen s.
The aim o his wo k was o design hyd ogels sui able o so con ac lenses (SCLs) wi h an
a ini y o ACV and VACV and ha can sus ainedly elease hese d ugs on he ocula su ace
du ing daily wea ing (Figu e 1). Among he p oposed p ocedu es o endow he SCLs wi h an
a ini y o speci ic molecules, he c ea ion o a i icial ecep o s using he molecula imp in ing
Acyclo i
Valacyclo i
S uc u al monome
Func ional monome
Figu e 1. S uc u e o acyclo i (ACV) and alacyclo i (VACV) and wo k low o he expe imen s.
Polyme s 2020,12, 2026 3 o 19
The aim o his wo k was o design hyd ogels sui able o so con ac lenses (SCLs) wi h an
a ini y o ACV and VACV and ha can sus ainedly elease hese d ugs on he ocula su ace du ing
daily wea ing (Figu e 1). Among he p oposed p ocedu es o endow he SCLs wi h an a ini y o
speci ic molecules, he c ea ion o a i icial ecep o s using he molecula imp in ing echnique s ands
ou [
22
–
24
]. This echnique equi es inco po a ing he d ug in o he monome s mix u e so ha
he monome s can ea ange acco ding o hei a ini y. This ea angemen becomes pe manen du ing
polyme iza ion. The emo al o he empla e molecules gene a es ca i ies wi h he mos app op ia e
size and chemical g oups o hos he d ug o in e es again [
25
]. The molecula imp in ing app oach has
been success ully applied o de elop SCLs loaded wi h an iglaucoma [
22
,
26
], an ialle gic [
27
,
28
], and
an imic obial [
29
,
30
] d ugs, among o he s, and he apeu ic agen s ha may inc ease ocula com o [
31
]
and e en add ess he managemen o diabe ic eyes [32,33], bu no wi h an i i al d ugs ye .
To ca y ou he wo k, unc ional monome s sui able o in e ac ion wi h he an i i al d ugs
we e i s sc eened using compu a ional modeling, a echnique ha has been shown o be e icien
o sa e ime and ma e ials in he de elopmen o imp in ed ma e ials [
34
]. Me hac ylic acid (MAA)
showed a highe a ini y o he d ugs han he s uc u al monome 2-hyd oxye hyl me hac yla e
(HEMA) and o he unc ional monome s. MAA may in e ac wi h he side chain o VACV h ough no
only hyd ogen bonding wi h he ing (as in he case o ACV) bu also elec os a ic in e ac ions wi h
he amino g oup o he aline chain. Hyd ogels we e p epa ed wi h a ious con en s in he unc ional
monome in he p esence (imp in ed) and absence (non-imp in ed) o each d ug. The hyd ogels
we e cha ac e ized in e ms o swelling, ligh ansmission, mechanical p ope ies, eye compa ibili y
(hen’s egg es on cho ioallan oic memb ane (HET-CAM) assay), and capabili y o load and elease
he an i i al d ugs. The s abili y o VACV loading solu ion/hyd ogels unde s eam hea s e iliza ion
and subsequen d ug elease was in es iga ed. Finally, he pe meabili y h ough bo ine and po cine
co nea and scle a o he d ug eleased om he hyd ogels was e alua ed (Figu e 1).
2. Ma e ials and Me hods
2.1. Ma e ials
Acyclo i (ACV; MW 225.21 g/mol; solubili y in wa e 1.02 mg/mL [
35
]) was pu chased om
Fa malabo (Canosa di Puglia, I aly); alacyclo i hyd ochlo ide (VACV; MW 360.80 g/mol; solubili y in
wa e 174 mg/mL [
36
]) was om Ac os O ganics (Geel, Belgium); 2,2
0
-azo-bis(isobu y oni ile) (AIBN),
dichlo odime hylsilane, e hylene glycol dime hac yla e (EGDMA), and me hac ylic acid (MAA) we e
om Sigma-Ald ich (S einheim, Ge many); e hanol absolu e and NaOH we e om VWR (Leu en,
Belgium); 2-hyd oxye hyl me hac yla e (HEMA) was om Me ck (Da ms ad , Ge many); ace ic acid
and NaCl we e om Scha lau (Sen mena , Spain); and me hanol was om Fishe (Loughbo ough,
UK). Ul apu e wa e ( esis i i y >18 M
Ω·
cm) was ob ained by e e se osmosis (MilliQ
®
, Millipo e,
Mad id, Spain). Simula ed lac imal luid (SLF) was p epa ed wi h he ollowing composi ion: 6.78 g/L
NaCl, 2.18 g/L NaHCO
3
, 1.38 g/L KCl, and 0.084 g/L CaCl
2·
2H
2
O wi h pH 7.5. Ca bona e bu e pH 7.2
was p epa ed by mixing bu e solu ion A (6.2 g/L NaCl, 0.355 g/L KCl, 0.1 g/L NaH
2
PO
4·
H
2
O, and
2.45 g/L NaHCO3) and bu e solu ion B (0.115 g/L CaCl2and 0.155 g/L MgCl2·6H2O).
2.2. Compu a ional Modeling
A p elimina y s udy was ca ied ou using compu e modeling o elucida e in e ac ions be ween
he d ugs o be s udied (ACV and VACV) and unc ional monome s used in he syn hesis o hyd ogels.
The es ed monome s we e ac ylamide (AAm), 2-aminoe hyl me hac yla e hyd ochlo ide (AEMA),
N-(3-aminop opyl) me hac ylamide hyd ochlo ide (APMA), e hylene glycol phenyl e he me hac yla e
(EGPEM), bu oxye hyl me hac yla e (BEM), hyd oxye hyl me hac yla e (HEMA), and me hac ylic
acid (MAA). The 3D s uc u e o he unc ional monome s and ACV and VACV was aken om
he PubChem da abase [
37
]. The SDF iles we e ans o med o PDB iles using OpenBabel . 2.4.1
so wa e [
38
]. The Au odock Tools . 4.2.6 so wa e was used o calcula e molecula docking. In all
Polyme s 2020,12, 2026 4 o 19
cases, he g id was gene a ed wi h de aul se ings a ound he monome and he d ug, he smalles
con o ma ion was used, and he docking was pe o med using he Lama ckian Gene ic Algo i hm [
39
].
Es ima ed ee ene gy o binding (
∆
G
binding
) and dissocia ion cons an (Ki) alues we e ob ained.
Au odock used a semi-empi ical o ce ield o e alua e he binding in wo s eps. The ligand (e.g.,
monome ) and ecep o (e.g., d ug) s a ed in an unbound con o ma ion. Fi s , he in amolecula
ene ge ics we e es ima ed o he ansi ion om hese unbound s a es o he con o ma ion in he bound
s a e. Second, he in e molecula ene ge ics o combining he ligand and ecep o we e es ima ed.
Ene gies o dispe sion/ epulsion, hyd ogen bonding, elec os a ics, and desol a ion we e e alua ed
as desc ibed in he use guide [
40
]. The dissocia ion cons an o he complex (Ki), also known as
he inhibi ion cons an , was es ima ed as ollows:
Ki =e(∆Gbinding/(RT)) (1)
In his equa ion, Ris he gas cons an and T he absolu e empe a u e in Kel in.
2.3. Syn hesis o Imp in ed and Non-Imp in ed Hyd ogels
Di e en mix u es o monome s we e p epa ed as shown in Table 1. The componen s we e
added o ials and mixed a oom empe a u e and unde magne ic agi a ion (300 pm) un il hey
we e comple ely dissol ed. Finally, he ini ia o (AIBN) was added, and he solu ions we e s i ed
o 15 min mo e. The solu ions we e injec ed, wi h a needle and sy inge, in o p e-assembled molds,
consis ing o wo p e- ea ed glass pla es (12
×
14 cm) sepa a ed by a 0.45 mm- hick silicone ame.
A e p e- ea men o he glass pla es wi h dichlo odime hylsilane, he pla es we e le o d y in a hood
o 1 h, ho oughly washed wi h e hanol, insed wi h wa e , and d ied in an o en a 70
◦
C o 1 h be o e
being assembled. Polyme iza ion o he monome s inside he molds was ca ied ou o 12 h a 50
◦
C
and hen o a u he 24 h a 70 ◦C. All hyd ogel composi ions we e p epa ed in iplica e.
Table 1.
Composi ion o he hyd ogels (NIP: Non-imp in ed hyd ogels, MIP: Imp in ed
hyd ogels). Final e hylene glycol dime hac yla e (EGDMA), me hac ylic acid (MAA), and
2,20-azo-bis(isobu y oni ile) (AIBN) concen a ions we e 8, 200 and 10 mM, espec i ely.
Hyd ogel HEMA
(mL)
EGDMA
(µL)
MAA
(mL)
ACV
(mg)
VACV
(mg)
AIBN
(mg)
NIP 5 7.55 0 0 0 8.21
NIP200 5 7.55 0.084 0 0 8.21
MIPACV 5 7.55 0 45 0 8.21
MIPA1:5 5 7.55 0.084 45 0 8.21
MIPA1:10 5 7.55 0.084 23 0 8.21
MIPA1:15 5 7.55 0.084 15 0 8.21
MIPVACV 5 7.55 0 0 25 8.21
MIPV1:6 5 7.55 0.084 0 50 8.21
MIPV1:12 5 7.55 0.084 0 25 8.21
MIPV1:32 5 7.55 0.084 0 10 8.21
2.4. D ug Remo al
A e polyme iza ion, each hyd ogel shee was imme sed in 500 mL o boiling wa e o 15 min in
o de o emo e un eac ed monome s and empla e d ugs and acili a e he cu ing in o discs (10 mm
in diame e ). Fu he washing was hen pe o med, excep o a ew discs o each ype o hyd ogel,
which we e ese ed o he di ec d ug elease es (as explained in Sec ion 2.5). Fo he washing,
he discs we e imme sed in wa e , unde magne ic agi a ion (300 pm) and a oom empe a u e.
The medium was eplaced e e y 24 h un il no signal was de ec ed in he ange o 190–800 nm
(UV-Vis spec opho ome e , Agilen 8534, Waldb onn, Ge many). When no spec opho ome ic signal
was de ec ed, he hyd ogels we e d ied in an o en a 70
◦
C o 24 h and s o ed p o ec ed om
Polyme s 2020,12, 2026 5 o 19
ligh and humidi y. In pa allel, he amoun o ACV (MIP
ACV
, MIP
A1:5
, MIP
A1:10
, and MIP
A1:15
) and
VACV (MIP
VACV
, MIP
V1:6
, MIP
V1:12
, and MIP
V1:32
) emo ed in each washing s ep was moni o ed
spec opho ome ically a 252 and 253 nm, espec i ely.
2.5. Di ec D ug Release Tes om Boiled Hyd ogels
A e he boiling s ep, h ee discs o each ype o hyd ogel we e indi idually placed in ials
con aining 5 mL o SLF and kep unde oscilla ing agi a ion (300 pm) a 35
◦
C. A p ese imes
(0.5, 1, 2, 6, and 24 h), 3 mL o medium was emo ed and he abso bance was measu ed a 252 nm
(ACV) and 253 nm (VACV) (UV-Vis spec opho ome e , Agilen 8453, Waldb onn, Ge many), e u ning
he samples o he elease ial. The expe imen s we e ca ied ou in iplica e. The amoun s o d ug
eleased we e calcula ed using p e iously p epa ed calib a ion cu es and e e ed o he uni o mass
o he d y disc. The calib a ion cu es we e p epa ed by dissol ing ACV (30
µ
g/mL) in e hanol:wa e
(50:50, / ) mix u e, and VACV (50
µ
g/mL) in wa e . Dilu ions o 2, 3, 5, 10, 15, 20, 25, and 30
µ
g/mL
we e made o ACV, and 1.25, 2.5, 5, 10, 15, 20, 25, 30, 40, and 50
µ
g/mL o VACV. The calib a ion cu es
we e alida ed o abso bances eco ded a 252 and 253 nm, espec i ely (UV-Vis spec opho ome e ,
Agilen 8453, Waldb onn, Ge many).
2.6. D ug Loading and Release
ACV loading was es ed, in iplica e, on he hyd ogels NIP, NIP
200
, MIP
ACV
, MIP
A1:5
, MIP
A1:10
,
and MIP
A1:15
. Each hyd ogel disc (app ox. 40 mg) p e iously washed and d ied was placed in a ube
wi h 5 o 15 mL o ACV aq. solu ion (0.3 mg/mL) and kep unde oscilla ing agi a ion (300 pm), a
oom empe a u e (23–25
◦
C), o 4 days. The abso bance o he solu ion was moni o ed a 252 nm
(UV-Vis spec opho ome e , Agilen 8453, Waldb onn, Ge many) by aking aliquo s o 0.2 mL and
dilu ing o 5 mL wi h e hanol:wa e mix u e (50:50, / ) (i.e., 1:25 dilu ion). The amoun o d ug loaded
was es ima ed by he di e ence be ween he ini ial and inal amoun o d ug in solu ion calcula ed
using he p e iously p epa ed calib a ion cu e, and e e ed o he uni o mass o he d y disc.
VACV loading was e alua ed, in iplica e, on he hyd ogels NIP, NIP
200
, MIP
VACV
, MIP
V1:6
,
MIP
V1:12
, and MIP
V1:32
. Each hyd ogel disc (app ox. 40 mg) p e iously washed and d ied was placed
in a ube wi h 5 mL o VACV (0.3 mg/mL) solu ion in 0.1 mM NaOH medium (pH 6.6). The loading
ubes we e kep unde he same condi ions o agi a ion, empe a u e, and ime as o he ACV loading.
The abso bance o he medium was moni o ed spec opho ome ically a 253 nm.
The d ug ne wo k/wa e pa i ion coe icien (K
N/W
) was calcula ed o each hyd ogel om he o al
amoun o d ug loaded using he ollowing equa ion
Loading ( o al)=VS+KN/W∗Vp
WP
∗C0(2)
whe e V
S
is he olume o wa e abso bed by he hyd ogel (mL), V
p
he olume o d y polyme (mL),
W
p
he weigh o he d y hyd ogel (g), and C
0
he concen a ion o d ug in he loading solu ion (g/mL).
The loaded discs we e emo ed om he ubes and insed wi h wa e . The su ace wa e was
emo ed wi h il e pape , and he discs we e hen immedia ely placed in elease ubes wi h 15 and
10 mL o SLF ( o ACV and VACV, espec i ely) unde oscilla ing agi a ion (300 pm) and a 35
◦
C.
The elease kine ics was e alua ed o 24 h. Samples o he medium we e pe iodically aken and
analyzed ollowing he same p o ocol as in Sec ion 2.5.
Feasibili y o he simul aneous loading and s e iliza ion o he hyd ogels was also in es iga ed.
D ied NIP, NIP
200
, MIP
VACV
, MIP
V1:6
, MIP
V1:12
, and MIP
V1:32
hyd ogels we e placed in ials con aining
5 mL o VACV (0.3 mg/mL) solu ion in 0.1 mM NaOH medium (pH 6.6) and kep o 12 h a oom
empe a u e. Then, he ials we e s eam hea s e ilized (au ocla e, 121
◦
C, 30 min) and s o ed a oom
empe a u e o 24 h wi hou shaking. VACV solu ions wi hou hyd ogels we e p ocessed as con ols.
D ug loading and elease p o iles we e eco ded as explained abo e. The s e iliza ion es s we e
ca ied ou in iplica e o each hyd ogel and VACV solu ion and epea ed in wo independen uns.

Polyme s 2020,12, 2026 6 o 19
2.7. Sol en Up ake
The up ake o wa e and SLF was moni o ed eco ding he inc ease in weigh o d ied discs
a e being imme sed in 4 mL o he co esponding medium a oom empe a u e (23–25
◦
C). A
p ede e mined imes (0.5, 1, 2, 4, 8, and 24 h), each disc was aken om he ial, excess wa e was
emo ed wi h blo ing pape , and he weigh eco ded. The discs we e immedia ely e u ned o
he ials. The sol en up ake was calcula ed as ollows:
Sol en up ake (%)=W −W0
W0
∗100 (3)
whe e W0and W ep esen he weigh o he d ied and swollen hyd ogel, espec i ely.
2.8. Ligh T ansmission
The ligh ansmi ance (%) o discs swollen in SLF was measu ed in a spec opho ome e (Agilen
Ca y 60 UV-Vis, Waldb onn, Ge many) in iplica e om 200 o 800 nm.
2.9. Mechanical P ope ies
The mechanical p ope ies o NIP, NIP
200
, MIP
VACV
, MIP
V1:6
, MIP
V1:12
, and MIP
V1:32
hyd ogels
swollen in wa e we e es ed in iplica e a oom empe a u e (23–25
◦
C). Each hyd ogel was cu in o 16
×
9 mm s ips and a ached o he uppe and lowe clamps, wi h a 7 mm gap, on a TA.XT Plus Tex u e
Analyze (S able Mic o Sys ems L d., Su ey, UK), equipped wi h a 5 kg load cell. The c osshead speed
applied o eco d he s ess–s ain plo s was 0.1 mm/s. The Young’s modulus (E) was calcula ed as
he slope o he s aigh -line pa o enginee ing s ess ( o ce pe c oss-sec ional a ea, N/mm
2
) e sus
he enginee ing s ain (change in ac i e leng h di ided by o iginal leng h, mm/mm) [
41
,
42
] as ollows:
E=
F
A0
∆L
L0
(4)
2.10. HET-CAM Tes
The hen’s egg es on cho ioallan oic memb ane (HET-CAM) was pe o med by incuba ing
e ilized hen eggs (50–60 g) a 37
◦
C and 60% RH o 9 days. On he nin h day o incuba ion, a ci cula
cu was made on he op o he egg o app oxima ely 1 cm diame e wi h a o a y saw (D emel 300,
B eda, The Ne he lands). The shell was emo ed, and he inne memb ane was mois ened wi h 0.9%
NaCl o 30 min ( ime du ing which he egg emained inside he clima ic chambe ). The memb ane was
hen emo ed o expose he cho ioallan oic memb ane (CAM) [
33
]. The es was pe o med by placing
in each CAM a hyd ogel disc p e iously soaked o 4 days in d ug loading solu ion. Aqueous solu ions
o NaOH 0.1 N and NaCl 0.9% (300
µ
L) we e used as nega i e and posi i e con ols, espec i ely.
The blood essels we e obse ed unde whi e ligh o 5 min, o de ec possible bleeding, ascula lysis,
o coagula ion. All es s we e pe o med in iplica e and he i i a ion sco e calcula ed as epo ed
p e iously [33].
2.11. Co nea and Scle a Pe meabili y Tes s
F esh bo ine and po cine eyes we e collec ed om a local slaugh e house and anspo ed
acco ding o he Bo ine Co neal Opaci y/Pe meabili y (BCOP) es p o ocol [
33
,
43
]. Du ing anspo ,
he eyes we e kep imme sed in PBS wi h added an ibio ics (penicillin 100 IU/mL and s ep omycin
100
µ
g/mL), in an ice ba h. Co neas and scle as we e isola ed using a scalpel. The issues we e washed
wi h 0.9% NaCl and moun ed in e ical di usion cells (F anz cells). To balance he issues, he dono
and ecep o chambe s we e illed wi h ca bona e bu e pH 7.2 and placed in a ba h a 37
◦
C, wi h
magne ic s i ing, o 30 min. A e ha ime, he con en o he dono chambe was emo ed and
he co neas and scle as we e exposed o VACV-loaded NIP and MIP
V1:12
discs (loaded as in Sec ion 2.6).
Polyme s 2020,12, 2026 7 o 19
The discs we e co e ed wi h 2 mL o 0.9% NaCl. In pa allel, co neas and scle as we e exposed o 2 mL
o a VACV solu ion (100
µ
g/mL in NaOH 0.1 mM; pH 6.6) as a con ol, o 6 h. The dono chambe s
we e co e ed wi h pa a ilm o a oid e apo a ion. Samples (1 mL) o he ecep o medium we e aken
a 0.5, 1, 2, 3, 4, 5, and 6 h, and eplaced wi h ca bona e bu e pH 7.2 aking ca e o p e en ing bubbles
o ma ion in he di usion cell.
The amoun o VACV pe mea ed in o he ecep o chambe was quan i ied by HPLC (Au osample
Wa e s 717, Wa e s Con olle 600, Pho odiode De ec o 996, Mil o d, MA, USA), equipped wi h
a C18 column (Wa e s Symme y C18, 5
µ
m, 4.6
×
250 mm) and ope a ed wi h he Empowe 2
so wa e. The mobile phase consis ed o ace ic acid (1:1000): me hanol (90:10 / ) wi h a low a e
o 1 mL/min. The injec ion olume was 50
µ
L and he column was kep a 30
◦
C. The calib a ion
was pe o med wi h s anda d solu ions o ACV and VACV (6.25–0.19
µ
g/mL) in ca bona e bu e pH
7.2 [
44
], and he abso bance was quan i ied a 251 nm. Re en ion imes we e ~3 min o VACV and
4.8 min o ACV ( ypical HPLC ch oma og ams a e shown in Figu e S1, Suppo ing In o ma ion).
The accumula ed amoun s o d ug pe mea ed we e calcula ed om he sum o VACV and ACV
peaks [
20
]. The s eady-s a e low (J) and he ime delay (
lag
) we e ob ained om he slope and
x-in e cep , espec i ely, o he linea eg ession o he accumula ed amoun o d ug pe mea ed pe
a ea s. ime [
45
]. A e 6 h o he es , aliquo s o he liquid emaining a he dono chambe we e
aken o u he analysis. The coe icien s o pe meabili y o he d ug h ough he co nea and scle a
we e calcula ed as he a io o J o he concen a ion o d ug in he dono chambe [45].
The co neas/scle as we e also emo ed om he di usion cells a e 6 h es , insed wi h 0.9% NaCl,
and imme sed in 3 mL o an e hanol:wa e mix u e (50:50 / ) o e nigh . They we e hen sonica ed o
99 min a 37
◦
C, cen i uged (1000 pm, 5 min, 25
◦
C), il e ed, and e-cen i uged (14,000 pm, 20 min,
25
◦
C) [
46
]. The d ug ex ac ed om he co neas/scle as was quan i ied by HPLC as explained abo e.
2.12. S a is ical Analysis
The e ec s o hyd ogel composi ion on d ug loading and pe meabili y h ough po cine and bo ine
issues we e analyzed using ANOVA and he mul iple ange es (S a g aphics Cen u ion XVII, S a
Poin Technologies Inc., Wa en on, VA, USA).
3. Resul s and Discussion
3.1. Compu a ional Modeling
Compu a ional modeling is a e sa ile ool o he i s sc eening o unc ional monome s sui able
o p epa ing imp in ed hyd ogels [
34
,
47
,
48
]. Al hough he condi ions du ing polyme iza ion canno
be p ecisely esembled, compu a ional modeling has been shown o be use ul o iden i y he monome s
wi h a high a ini y o he empla e d ug, which, in u n, may endow he imp in ed hyd ogels wi h
high ebinding and con olled elease pe o mances [
48
,
49
]. HEMA, as a main s uc u al monome
o SCLs, and six unc ional monome s bea ing di e en chemical moie ies (amido, amine, phenyl,
bu yl, and ac ylic acid) we e sc eened ega ding hei in e ac ions wi h ACV and VACV. Resul s o
compu a ional modeling a e summa ized in Figu e 2.
High nega i e alues o
∆
G
binding
indica ed a o able binding in e ac ions be ween he d ug and
he monome . The lowe he alue o Ki, he lowe he likelihood o complex disassembly. Conside ing
bo h
∆
G
binding
and Ki, he in e ac ion o ACV and VACV wi h MAA was p edic ed o be mo e a o able
han wi h o he monome s (Figu e 2). In e es ingly, MAA may in e ac wi h VACV h ough hyd ogen
bonds wi h he amino g oups in he a oma ic ing (as in he case o ACV) and also wi h he p ima y
amine o he aline side chain. The pKa o he amino g oup o aline has been es ima ed o be in
he 9.1–9.6 ange [
50
]. Thus, in he aqueous medium used o load he hyd ogels and in he lac imal
luid, his amino g oup is expec ed o emain p o ona ed. This opens he possibili y o he ac ha
he MAA me s in he hyd ogels may eadily in e ac wi h VACV h ough elec os a ic in e ac ions.
The e o e, MAA was chosen as he unc ional monome o p epa e he hyd ogels.
Polyme s 2020,12, 2026 8 o 19
Polyme s 2020, 12, x FOR PEER REVIEW 8 o 19
Figu e 2. Compu a ional modeling esul s o he in e ac ion o acyclo i (ACV) wi h he monome s
2-hyd oxye hyl me hac yla e (HEMA), ac ylamide (AAm), 2-aminoe hyl me hac yla e
hyd ochlo ide (AEMA), N-(3-aminop opyl) me hac ylamide hyd ochlo ide (APMA), bu oxye hyl
me hac yla e (BEM), e hylene glycol phenyl e he me hac yla e (EGPEM), and me hac ylic acid
(MAA); and he in e ac ion o alacyclo i (VACV) wi h 2-hyd oxye hyl me hac yla e (HEMA) and
me hac ylic acid (MAA).
3.2. Syn hesis o Hyd ogels and D ug Remo al
Imp in ed and non-imp in ed hyd ogels we e syn hesized combining HEMA wi h MAA as he
unc ional monome . The d ug was added a di e en le els, in ascending mole a ios o ACV:MAA
(1:5, 1:10, and 1:15 mol/mol) and VACV:MAA (1:6, 1:12, and 1:32 mol/mol), as explained in Table 1. I
should be no ed ha he o al con en in MAA was ixed, and only he con en in he empla e d ug
a ied. The a io VACV:MAA was lowe han ha o ACV:MAA because o he addi ional binding
poin s ha VACV may es ablish wi h MAA acco ding o he compu a ional s udy. The use o MAA
as comonome should enhance d ug–hyd ogel in e ac ions h ough hyd ogen bonding o he ac ylic
acid g oup wi h he ings o he d ugs (ACV and VACV) and elec os a ic in e ac ions wi h he
Figu e 2.
Compu a ional modeling esul s o he in e ac ion o acyclo i (ACV) wi h he monome s
2-hyd oxye hyl me hac yla e (HEMA), ac ylamide (AAm), 2-aminoe hyl me hac yla e hyd ochlo ide
(AEMA), N-(3-aminop opyl) me hac ylamide hyd ochlo ide (APMA), bu oxye hyl me hac yla e (BEM),
e hylene glycol phenyl e he me hac yla e (EGPEM), and me hac ylic acid (MAA); and he in e ac ion
o alacyclo i (VACV) wi h 2-hyd oxye hyl me hac yla e (HEMA) and me hac ylic acid (MAA).
3.2. Syn hesis o Hyd ogels and D ug Remo al
Imp in ed and non-imp in ed hyd ogels we e syn hesized combining HEMA wi h MAA as
he unc ional monome . The d ug was added a di e en le els, in ascending mole a ios o ACV:MAA
(1:5, 1:10, and 1:15 mol/mol) and VACV:MAA (1:6, 1:12, and 1:32 mol/mol), as explained in Table 1. I
should be no ed ha he o al con en in MAA was ixed, and only he con en in he empla e d ug
a ied. The a io VACV:MAA was lowe han ha o ACV:MAA because o he addi ional binding
poin s ha VACV may es ablish wi h MAA acco ding o he compu a ional s udy. The use o MAA
as comonome should enhance d ug–hyd ogel in e ac ions h ough hyd ogen bonding o he ac ylic
acid g oup wi h he ings o he d ugs (ACV and VACV) and elec os a ic in e ac ions wi h he VACV
side chain. The highes d ug:MAA mole a io was limi ed by he poo solubili y o he d ugs in
Polyme s 2020,12, 2026 9 o 19
he monome s solu ion, bu also conside ing ha 1:4 o 1:6 mole a ios ha e been commonly epo ed
as adequa e o c ea e imp in ed ca i ies [
34
,
51
]. Addi ionally, non-imp in ed hyd ogels p epa ed wi h
he same con en in MAA (NIP
200
) and imp in ed hyd ogels wi hou MAA (MIP
ACV
and MIP
VACV
)
we e syn hesized unde he same condi ions in o de o elucida e he ole o he unc ional monome
and o he d ug empla e, espec i ely.
Monome s and empla e d ug molecules we e easily de ec ed in he washing medium. Monome s
mainly abso bed a wa eleng hs below 220 nm, while ACV and VACV abso bed a 252–253 nm. Bo h
peaks we e clea ly eco ded in he i s washing solu ion a e boiling. VACV was easily emo ed om
he VACV-imp in ed hyd ogels du ing he washing p ocess in boiling wa e (Figu e 3a). Elu ion o
esidual monome s caused mino in e e ences in he quan i ica ion o he amoun s o d ug ex ac ed.
By con as , he amoun s o ACV emo ed om he hyd ogels syn hesized using ACV as a empla e
we e lowe han he amoun s added du ing syn hesis (Figu e 3a). This could be due o he limi ed
solubili y o he ACV in aqueous medium, which may a o hyd ophobic associa ion wi h he hyd ogel
polyme backbone.
Polyme s 2020, 12, x FOR PEER REVIEW 9 o 19
VACV side chain. The highes d ug:MAA mole a io was limi ed by he poo solubili y o he d ugs
in he monome s solu ion, bu also conside ing ha 1:4 o 1:6 mole a ios ha e been commonly
epo ed as adequa e o c ea e imp in ed ca i ies [34,51]. Addi ionally, non-imp in ed hyd ogels
p epa ed wi h he same con en in MAA (NIP200) and imp in ed hyd ogels wi hou MAA (MIPACV
and MIPVACV) we e syn hesized unde he same condi ions in o de o elucida e he ole o he
unc ional monome and o he d ug empla e, espec i ely.
Monome s and empla e d ug molecules we e easily de ec ed in he washing medium.
Monome s mainly abso bed a wa eleng hs below 220 nm, while ACV and VACV abso bed a 252–
253 nm. Bo h peaks we e clea ly eco ded in he i s washing solu ion a e boiling. VACV was
easily emo ed om he VACV-imp in ed hyd ogels du ing he washing p ocess in boiling wa e
(Figu e 3a). Elu ion o esidual monome s caused mino in e e ences in he quan i ica ion o he
amoun s o d ug ex ac ed. By con as , he amoun s o ACV emo ed om he hyd ogels
syn hesized using ACV as a empla e we e lowe han he amoun s added du ing syn hesis (Figu e
3a). This could be due o he limi ed solubili y o he ACV in aqueous medium, which may a o
hyd ophobic associa ion wi h he hyd ogel polyme backbone.
Figu e 3. (a) Amoun s o ACV and VACV emo ed du ing washing in boiling wa e and subsequen
washings om each o he hyd ogels es ed (codes as in Table 1), and (b) elease p o iles o ACV and
VACV om he imp in ed discs ha we e p e iously boiled in wa e (15 min) and d ied o cons an
weigh . The d ug eleased co esponds o ha used as a empla e du ing syn hesis.
3.3. Di ec D ug Release Tes om Boiled Hyd ogels
A di ec elease in SLF was ca ied ou o hyd ogels a e boiling (wi hou u he washing) o
he i s sc eening o hei abili y o elease he emaining ACV and VACV empla e in a sus ained
way (Figu e 3b). The hyd ogels polyme ized using ACV as a empla e (MIPACV, MIPA1:5, MIPA1:10,
MIPA1:15) eleased abou 0.20 mg o d ug pe g am o disc. The sum o he amoun eleased plus he
amoun al eady emo ed du ing boiling was s ill lowe han he o al amoun added du ing
syn hesis (45, 45, 23, and 15 mg o ACV, espec i ely), and p ac ically no di e ences we e obse ed
be ween he di e en ypes o hyd ogel. This inding poin ed o ACV empla e en apmen in he
polyme ne wo k, a o ed by he hyd ophobici y o he d ug. By con as , he hyd ogels
polyme ized using VACV as he empla e (MIPVACV, MIPV1:6, MIPV1:12, MIPV1:32) eleased he small
amoun o emnan VACV in a sus ained way o 24 h. The mass balance o VACV con i med ha
he molecules used as he empla e we e mos ly emo ed du ing he boiling p ocess. A simila
elease expe imen was pe o med wi h non-imp in ing hyd ogels and, as expec ed, no signal was
eco ded a he wa eleng h used o d ug quan i ica ion.
ACV/VACV emo ed (mg/g)
0
10
20
30
40
50 Boiling
Subsequen washings
Time (h)
0 4 8 12 16 20 24
ACV/VACV emo ed (mg/g)
0.0
0.2
0.4
0.6
0.8
1.0
MIPACV
MIPA1:5
MIPA1:10
MIPA1:15
MIPVACV
MIPV1:6
MIPV1:12
MIPV1:32
MIPACV
MIPA1:5
MIPA1:10
MIPA1:15
MIPVACV
MIPV1:6
MIPV1:12
MIPV1:32
(a) (b)
Figu e 3.
(
a
) Amoun s o ACV and VACV emo ed du ing washing in boiling wa e and subsequen
washings om each o he hyd ogels es ed (codes as in Table 1), and (
b
) elease p o iles o ACV and
VACV om he imp in ed discs ha we e p e iously boiled in wa e (15 min) and d ied o cons an
weigh . The d ug eleased co esponds o ha used as a empla e du ing syn hesis.
3.3. Di ec D ug Release Tes om Boiled Hyd ogels
A di ec elease in SLF was ca ied ou o hyd ogels a e boiling (wi hou u he washing) o
he i s sc eening o hei abili y o elease he emaining ACV and VACV empla e in a sus ained way
(Figu e 3b). The hyd ogels polyme ized using ACV as a empla e (MIP
ACV
, MIP
A1:5
, MIP
A1:10
, MIP
A1:15
)
eleased abou 0.20 mg o d ug pe g am o disc. The sum o he amoun eleased plus he amoun
al eady emo ed du ing boiling was s ill lowe han he o al amoun added du ing syn hesis (45, 45,
23, and 15 mg o ACV, espec i ely), and p ac ically no di e ences we e obse ed be ween he di e en
ypes o hyd ogel. This inding poin ed o ACV empla e en apmen in he polyme ne wo k, a o ed
by he hyd ophobici y o he d ug. By con as , he hyd ogels polyme ized using VACV as he empla e
(MIP
VACV
, MIP
V1:6
, MIP
V1:12
, MIP
V1:32
) eleased he small amoun o emnan VACV in a sus ained
way o 24 h. The mass balance o VACV con i med ha he molecules used as he empla e we e mos ly
emo ed du ing he boiling p ocess. A simila elease expe imen was pe o med wi h non-imp in ing
hyd ogels and, as expec ed, no signal was eco ded a he wa eleng h used o d ug quan i ica ion.
Polyme s 2020,12, 2026 16 o 19
4. Conclusions
Acyclo i and alacyclo i beha ed di e en ly when inco po a ed in o HEMA-based hyd ogels,
despi e hei simila chemical s uc u e and a p io i simila binding ene gy wi h MAA. The limi ed
solubili y o ACV in he monome mix u e oge he wi h unspeci ic hyd ophobic in e ac ions may
explain why ACV-imp in ed hyd ogels we e no e ec i e in e ms o d ug loading and elease.
Mo eo e , he small amoun o ACV loaded was no comple ely eleased in SLF, p obably because
o he hyd ophobici y o he d ug. By con as , he use o MAA as unc ional monome ema kably
inc eased he a ini y o he hyd ogels o VACV h ough elec os a ic in e ac ions be ween he ac ylic
acid g oup o he monome and he d ug la e al chain. The use o VACV as a empla e du ing
polyme iza ion acili a ed he a angemen o he polyme ne wo k, c ea ing speci ic ca i ies ha
con ibu ed o enhancing he a ini y o he d ug o he hyd ogel in subsequen loading. The deg ee o
swelling, ligh ansmission, and mechanical p ope ies showed common alues o daily wea con ac
lenses. In addi ion, no po en ial eye i i a ion was obse ed in he HET-CAM assay. VACV-imp in ed
hyd ogels can elease he d ug in a sus ained manne o 10 h, which is a common ime o wea ing
disposable SCLs. The apeu ically ele an amoun s accumula ed in he co nea. A he scle a le el,
VACV-loaded hyd ogels showed pe meabili y alues equi alen o hose achie ed wi h he aqueous
solu ion o he d ug. VACV pe meabili y h ough he scle a sugges s he possibili y o deli e y o
he pos e io segmen . The e o e, hyd ogels con aining MAA and imp in ed wi h VACV a e sui able
candida es o he p epa a ion o d ug-elu ing con ac lenses. As VACV does no wi hs and s eam hea
s e iliza ion, he scale-up may in ol e he au ocla ing o he hyd ogels i s and hen he packaging in
VACV solu ion p epa ed using s e ilizing il a ion.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h p://www.mdpi.com/2073-4360/12/9/2026/s1,
Figu e S1: Typical HPLC ch oma og ams o VACV and ACV s anda d solu ions and o a sample o he ecep o
medium du ing he pe meabili y es s. Figu e S2. HPLC ch oma og ams o he VACV loading solu ion be o e and
a e s eam hea s e iliza ion (au ocla e 121
◦
C, 30 min). Figu e S3. D ug (VACV+ACV) elease p o iles in SLF
om non-imp in ed and imp in ed hyd ogels ha we e loaded by soaking in VACV solu ion and s e ilized by
s eam hea s e iliza ion. The da a a e shown as accumula ed amoun s ob ained a e con e sion om abso bance
alues eco ded a 253 nm.
Au ho Con ibu ions:
Concep ualiza ion, A.C. and C.A.-L.; Me hodology, A.V.-G. and J.L.G.-A.; So wa e,
J.L.G.-A.; In es iga ion, A.V.-G. and C.A.-L.; Resou ces, A.C. and C.A.-L.; W i ing—o iginal d a p epa a ion,
A.V.-G. and C.A.-L.; W i ing— e iew and edi ing, A.C., J.L.G.-A. and C.A.-L.; Supe ision, A.C. and C.A.-L.;
unding acquisi ion, A.C. and C.A.-L. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This esea ch was unded by MINECO [SAF2017-83118-R] Spain, Agencia Es a al de In es igaci
ó
n (AEI)
Spain, Xun a de Galicia [ED431C 2016/008; AEMAT ED431E 2018/08] Spain, and FEDER.
Con lic s o In e es :
The au ho s decla e no con lic o in e es . The unde s had no ole in he design o he s udy;
in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip , o in he decision o publish
he esul s.
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