Strategies for ocular administration of antiviral and antioxidant agents

DOCTORAL THESIS
STRATEGIES FOR OCULAR
ADMINISTRATION OF ANTIVIRAL AND
ANTIOXIDANT AGENTS
Ángela Va ela Ga cía
INTERNATIONAL DOCTORAL SCHOOL
DOCTORAL PROGRAM IN DRUG RESEARCH AND DEVELOPMENT
SANTIAGO DE COMPOSTELA
2020
TESIS DOCTORAL
ESTRATEGIAS PARA LA
ADMINISTRACIÓN OCULAR DE
FÁRMACOS ANTIVIRALES Y
ANTIOXIDANTES
Ángela Va ela Ga cía
ESCUELA DE DOCTORADO INTERNACIONAL
PROGRAMA DE DOCTORADO EN INVESTIGACIÓN Y DESARROLLO DE
MEDICAMENTOS
SANTIAGO DE COMPOSTELA
2020
AUTHORIZATION OF THE THESIS SUPERVISORS
S a egies o ocula adminis a ion o an i i al and
an ioxidan agen s
P o . Ca men Al a ez Lo enzo
P o . Angel Conchei o Nine
REPORT:
Tha he p esen Thesis, co esponds o he wo k ca ied ou by Miss
Ángela Va ela Ga cía, unde ou supe ision, and ha we au ho ize i s
p esen a ion conside ing i ga he s he necessa y equi emen s o a icle
34 o he USC Doc o al S udies Regula ion, and ha as supe iso s o his
Thesis, i does no incu in he abs en ion causes es ablished by he law
40/2015
A San iago de Compos ela, on June 8
h
2020
P o . Ca men Al a ez Lo enzo P o . Angel Conchei o Nine

AUTORIZACIÓN DEL DIRECTOR / TUTOR DE LA TESIS
Es a egias pa a la adminis ación ocula de á macos
an i i ales y an ioxidan es
P o . Ca men Al a ez Lo enzo
P o . Angel Conchei o Nine
INFORMAN:
Que la p esen e Tesis, se co esponde con el abajo ealizado po Dña.
Ángela Va ela Ga cía, bajo nues a supe isión y au o izamos su
p esen ación
, conside ando
que eúne l o s
equisi os
exigidos en la
Regulación
de Es udios de
Doc o ado de la USC, y
que
como
di ec o es de és a
no incu e en las causas de
abs ención
es ablecidas e
n la Ley
40/2015.
En San iago de Compos ela, a 8 de Junio de 2020
P o . Ca men Al a ez Lo enzo P o . Angel Conchei o Nine
PhD CANDIDATE STATEMENT
S a egies o ocula adminis a ion o an i i al and
an ioxidan agen s
Miss Ángela Va ela Ga cía
I submi my Doc o al Thesis, ollowing he p ocedu e acco ding o he
Regula ion, s a ing ha :
1) This Thesis ga he s he esul s co esponding o my wo k.
2) When necessa y, explici men ion is gi en o he collabo a ions he
wo k may ha e had.
3) The p esen documen is he inal e sion submi ed o i s de ense
and coincides wi h he documen sen in elec onic o ma .
4) I con i m ha he Thesis does no incu in any plagia ism o any
o he au ho s o documen s submi ed by me o ob aining o he
deg ees.
A San iago de Compos ela, on June 8
h
2020
Sgd. Ángela Va ela Ga cía

AGRADECIMIENTOS
Cuando una e apa an impo an e de u ida llega a su in, es
ine i able echa la is a a ás pa a hace balance de odo lo posi i o
que ha ocu ido e in luido en u c ecimien o pe sonal y p o esional.
Son muchas las pe sonas que han apo ado algo du an e es e la go
ayec o, las cuales o ma án pa e de es a his o ia pa a siemp e. Po
ello, me gus a ía ag adece a odos los que de una o ma u o a han
es ado p esen es en ella.
A mis di ec o es de esis, los p o eso es Ca men Al a ez Lo enzo
y Angel Conchei o Nine. Es a é e e namen e ag adecida po habe me
dejado o ma pa e de ues o equipo. Me habéis enseñado que, con
es ue zo y pe se e ancia, odo se puede consegui . G acias po odo lo
ap endido.
A los p o eso es del Depa amen o de Fa macología, Fa macia y
Tecnología Fa macéu ica, José Luis Gómez Amoza, Ca los Ga cía
González, Ma iana Landín Pé ez, Loli To es López, Ca men
Remuñán López, y, en especial, a F ancisco O e o Espina , siemp e
dispues os a p es a ayuda en odo lo necesa io.
A los p o eso es Aisling Ní Annaidh, de School o Mechanical
and Ma e ials Enginee ing de la UCD de Dublín, y Lo enzo Pas ana,
del In e na ional Ibe ian Nano echnology Labo a o y de B aga, po
habe me dado la opo unidad de segui o mándome y de amplia mis
conocimien os desde o o pun o de is a.
A los ya doc o es, Ale, Sonia, Pa i, Isa y Luis, po da me la
bien enida a es a amilia y po ayuda a esol e siemp e las dudas de
una no a a. En especial a Fe , que pasó de se men o a amigo, po
habe pasado an as ho as explicándome y ayudándome, po odas las
isas y cab eos, y po el apoyo cons an e.
A mis colegas de más e , Lau a, Lucía, Ana y Diego, po habe
compa ido an os momen os, po esas cha las in ini as que nunca son
su icien e y po odo el apoyo mu uo.
Al “Team F an”, Vic o ia, And ea Conde, Xu xo, Rubén, Guille,
I ia, Ca los y And ea Luaces, po esa locu a con agiosa que, sin duda,
hace que el día a día sea mucho más que ag adable.
A mis compañe os de ba alla. A Ma ía, po que es oy con encida
de que sin i nada se ía igual, po que u gene osidad no iene lími es y
po habe o jado es a boni a amis ad que se á pa a siemp e. A Xián,
po odas las con e saciones, isas y llan os compa idos. A los
ecinos de a iba, Cla a, Vi i, Rebeca y Ana, po se an buenos
compañe os y po habe pasado jun os an os buenos momen os, así
como a Lo ena y a Helena. A las inco po aciones más ecien es, Ana
Filipa, Iago, Axel y Pa i po que aunque lle emos poco iempo jun os,
el compañe ismo y el buen ambien e que enemos es en pa e g acias a
oso os. A los que ya se han ido, Mi ian y Ma iano, po habe es ado
siemp e dispues os a ayuda sin duda lo. Y a los que es u ie on de
paso, pe o deja on huella, a Anna Paula, Ca ia y Ad ián, po que en
poco iempo habéis pasado a o ma pa e de es a g an amilia y, en
especial, a Claudia, po odos los buenos momen os compa idos y po
es a amis ad que nos une.
A And ea, po habe sido una compañe a de abajo excepcional,
po odos los buenos momen os pasados y po odos los que end án.
A Sole. Resul a complicado exp esa en pocas palab as lo
ag adecida que es oy po odo lo que haces. Je a, compañe a, men o a
y amiga, siemp e dispues a a ayuda en odo. Ha sido un place y un
p i ilegio habe podido compa i es a e apa de mi ida con igo.
A odos mis amigos y a mi amilia, en especial a Rocío y Vanesa,
mis “he manas” mayo es; ambién a Diana, Co es y So ía, po es a
siemp e ahí. A Ma cos, mi compañe o de ida, cuya paciencia in ini a
y apoyo incondicional hacen que pueda man ene me siemp e a lo e.
Po úl imo, a las pe sonas más impo an es de mi ida, Pila y
Rica do, mis pad es, ejemplos máximos de supe ación y alen ía, que
me han sabido enseña lo más alioso de la ida, nunca endi se y no
eme a las di icul ades. No hay palab as su icien es pa a ag adece
odo el apoyo ecibido en cada momen o, sin el cual nunca llega ía a
se la pe sona que hoy en día soy.
A odos, de co azón, mil g acias.
Index
Resumen ........................................................................................................ 3
1. In oduc ion ........................................................................................... 19
1.1. Ocula ana omy ................................................................................ 22
1.2. Eye-de ense mechanisms ................................................................. 26
1.2.1. Lac imal ilm .............................................................................. 27
1.2.2. Co neal ba ie ........................................................................... 28
1.2.3. Non-co neal ba ie s ................................................................... 29
1.2.4. Blood ocula ba ie s .................................................................. 30
1.3. Ocula d ug adminis a ion ............................................................ 31
1.4. Co nea diseases ................................................................................ 34
1.4.1. Ocula auma ............................................................................. 35
1.4.2. Degene a i e diso de s ............................................................... 36
1.4.3. In lamma o y diseases ................................................................ 38
1.4.4. In ec ious diseases ...................................................................... 39
1.4.5. P e en ion and ea men o co neal lesions: an ioxidan agen s 39
1.5. Ocula i al in ec ions ..................................................................... 41
1.5.1. T ea men o ocula i al in ec ions ........................................... 44
1.6. Micelles and con ac lenses as pla o ms o ocula deli e y ....... 46
1.6.1. Polyme ic micelles ..................................................................... 46
1.6.2. Con ac lenses ............................................................................. 50
1.7. Re e ences ......................................................................................... 58
2. Aims ........................................................................................................ 73
3. Polyme ic micelles o acyclo i ocula deli e y: o mula ion and
co nea and scle a pe meabili y ................................................................. 81
3.1. In oduc ion ...................................................................................... 81

3.2. Ma e ials and me hods ..................................................................... 86
3.2.1. Ma e ials ..................................................................................... 86
3.2.2. Micelles p epa a ion and cha ac e iza ion .................................. 87
3.2.3. Rheological beha io .................................................................. 87
3.2.4. Acyclo i solubiliza ion ............................................................. 88
3.2.5. Micelle s abili y agains dilu ion ................................................ 89
3.2.6. Co neal and scle a pe meabili y assay ........................................ 90
3.3. Resul s and Discussion ..................................................................... 91
3.3.1. Micelles p epa a ion and cha ac e iza ion .................................. 91
3.3.2. Rheological beha io .................................................................. 93
3.3.3. Acyclo i solubiliza ion ............................................................. 94
3.3.4. Micelle s abili y agains dilu ion ................................................ 99
3.3.5. Co nea and scle a pe meabili y assay ....................................... 100
3.4. Concluding ema ks ....................................................................... 105
3.5. Re e ences ....................................................................................... 106
4. Imp in ed hyd ogels o acyclo i and alacyclo i ocula
adminis a ion ........................................................................................... 115
4.1. In oduc ion .................................................................................... 115
4.2. Ma e ials and me hods ................................................................... 121
4.2.1. Ma e ials ................................................................................... 121
4.2.2. Compu a ional modeling .......................................................... 122
4.2.3. Syn hesis o imp in ed hyd ogels ............................................. 122
4.2.4. D ug emo al ............................................................................ 124
4.2.5. Di ec d ug elease es om boiled hyd ogels ......................... 125
4.2.6. D ug loading and elease om condi ioned hyd ogels ............. 126
4.2.7. Deg ee o swelling .................................................................... 127
4.2.8. Ligh ansmi ance ................................................................... 128
4.2.9. Mechanical p ope ies .............................................................. 128
4.2.10. HET-CAM es ................................................................... 129
4.2.11. Bo ine co neal and scle al pe meabili y es ...................... 129
4.3. Resul s and Discussion ................................................................... 131
4.3.1. Compu a ional modeling .......................................................... 131
4.3.2. Syn hesis o hyd ogels and d ug emo al ................................ 133
4.3.3. Di ec d ug elease es om boiled hyd ogels ........................ 134
4.3.4. D ug loading in washed hyd ogels ........................................... 136
4.3.5. D ug elease ............................................................................. 138
4.3.6. Hyd ogel cha ac e iza ion ........................................................ 140
4.3.7. HET-CAM es ......................................................................... 142
4.3.8. Bo ine co neal and scle al pe meabili y es ............................ 143
4.4. Concluding ema ks ...................................................................... 146
4.5. Re e ences ....................................................................................... 147
5. Cy osine- unc ionalized bioinspi ed hyd ogels o ocula deli e y o
an ioxidan ans e ulic acid ................................................................... 155
5.1. In oduc ion .................................................................................... 155
5.2. Ma e ials and me hods .................................................................. 161
5.2.1. Ma e ials ................................................................................... 161
5.2.2. Hyd ogel syn hesis ................................................................... 162
5.2.3. Func ionaliza ion wi h cy osine ............................................... 163
5.2.4. Hyd ogels cha ac e iza ion ....................................................... 164
5.2.5. TA loading ............................................................................... 166
5.2.6. TA elease ................................................................................ 166
5.2.7. HET-CAM es ......................................................................... 167
5.2.8. Cy ocompa ibili y assay ........................................................... 167
5.2.9. An ioxidan ac i i y .................................................................. 168
5.2.10. Co nea and scle a pene a ion and accumula ion ............... 169
5.3. Resul s and discussion ................................................................... 171
5.3.1. Hyd ogels syn hesis and cy osine g a ing ............................... 171
5.3.2. Swelling, ligh ansmission and mechanical p ope ies ........... 175
5.3.3. TA loading ................................................................................ 177
5.3.4. TA elease ................................................................................. 180
5.3.5. Biocompa ibili y ....................................................................... 182
5.3.6. An ioxidan ac i i y .................................................................. 183
5.3.7. Co nea and scle a pene a ion................................................... 185
5.4. Concluding ema ks ....................................................................... 189
5.5. Re e ences ....................................................................................... 190
6. Conclusions .......................................................................................... 201
7. Pa en .................................................................................................... 207
8. Abb e ia ions ....................................................................................... 239
Resumen
ÁNGELA VARELA GARCÍA
8
isión bo osa, en ojecimien o o lag imeo. La que a i is po he pes
simple (in ección e in lamación de la có nea) es la p incipal causa de
cegue a po in ección a ni el mundial. El aciclo i pene a en las
células in ec adas y compi e con los nucleósidos na u ales pa a
inco po a se al ADN y pode ac ua como e minado de cadena.
Después de ancla se, da luga a un mecanismo de inac i ación suicida,
pues o que el ADN e minado se une a la polime asa del ADN i al y
la inhibe de mane a i e e sible, impidiendo la eplicación del i us.
El a amien o ópico clásico equie e un pe iodo de aplicación
p olongado y da luga a e ec os secunda ios g a es. La adminis ación
o al se ca ac e iza po p esen a una biodisponibilidad in e io al 20%,
po lo que son necesa ias dosis muy al as du an e iempos
p olongados.
La hipó esis de es a p ime a e apa de la Tesis ue que la
encapsulación de aciclo i en nanomicelas polimé icas debe aumen a
la solubilidad del á maco y p omo e su acumulación en có nea y
escle a y el acceso a es uc u as más p o undas. Pa a lle a a cabo el
abajo, p ime o se hizo un ba ido de las p es aciones de los
copolíme os Soluplus y Solu ol. Se p epa a on dispe siones de cada
copolíme o cub iendo un amplio in e alo de concen aciones (1, 4, 8,
12, 16 y 20% p/p) en agua y ampón os a o (PBS) pH 7.4, y se e aluó
su amaño, po encial Z y compo amien o eológico.
Las p opiedades iscoelás icas se ca ac e iza on a pa i de
medidas de los módulos de almacenamien o (G’) y de pé dida (G’’) de

Resumen
9
las dispe siones de Soluplus y Solu ol al 12% y al 20%, en unción de
la empe a u a (15-40 °C). Las dispe siones de Solu ol mos a on un
compo amien o iscoso. En cambio, las dispe siones de Soluplus
mos a on inc emen os muy ma cados en G’ y G’’ a empe a u as
p óximas a la co po al. La capacidad de las nanomicelas pa a
solubiliza aciclo i se e aluó en agua, PBS pH 7.4 y luido lac imal
a i icial (FLS) pH 7.5. Se calcula on pa áme os de solubilidad como
la capacidad de solubilización mola , el coe icien e de epa o micela-
agua y la ene gía de solubilización lib e de Gibbs. Únicamen e las
micelas de Soluplus die on luga a inc emen os ele an es de la
solubilidad de aciclo i , mos ando una ele ada capacidad de
encapsulación y es abilidad en e a la dilución.
Finalmen e, se lle a on a cabo ensayos de pe meabilidad a a és
de có nea y escle a bo inas en células de di usión. En compa ación
con el aciclo i lib e en disolución, la encapsulación en micelas de
Soluplus dio luga a ma cados aumen os en la can idad de á maco
acumulada en ambos ejidos, así como en la can idad que pasa al
compa imen o ecep o . En suma, las micelas de Soluplus p esen a on
p opiedades isicoquímicas adecuadas pa a la adminis ación ocula de
aciclo i , mejo ando la pene ación del á maco a a és de có nea y
escle a.
2) Diseño de hid ogeles imp in ed pa a á macos an i i ales. Con
el in de p olonga la pe manencia del á maco en la supe icie ocula
más allá de lo que pe mi en las o mulaciones en nanomicelas, el
ÁNGELA VARELA GARCÍA
10
obje i o de la segunda e apa de la Tesis ue diseña hid ogeles álidos
pa a LC blandas con a inidad po aciclo i y alaciclo i . En e los
p ocedimien os p opues os pa a do a a los hid ogeles de a inidad po
moléculas especí icas, des aca la c eación de ecep o es a i iciales
u ilizando la écnica de moldeado molecula (molecula imp in ing).
Es a écnica equie e inco po a la sus ancia de in e és a la mezcla de
monóme os pa a que es os se eo denen en unción de su a inidad, y el
eo denamien o se haga pe manen e du an e la polime ización. La
emoción de las moléculas molde gene a ca idades con el amaño y
los g upos químicos más adecuados pa a aloja de nue o la sus ancia
de in e és.
La selección del ácido me ac ílico (MAA) como monóme o
uncional se hizo eniendo en cuen a que, además de se un monóme o
habi ual en la p epa ación de LC, en un es udio p elimina po
modelización compu acional mos ó capacidad pa a in e acciona
an o con el anillo de aciclo i y alaciclo i como con el g upo amino
de la cadena la e al alaciclo i .
Se sin e iza on hid ogeles mezclando HEMA con MAA como
monóme o uncional, y se añadió aciclo i o alaciclo i en dis in as
p opo ciones. T as la polime ización, se con i mó la ex acción del
á maco du an e los la ados median e espec o o ome ía UV. Con
algunos hid ogeles se lle ó a cabo un es udio de cesión di ec a del
á maco (sin la ado p e io), pa a de e mina la capacidad de con ol
de la cesión del á maco inco po ado du an e la polime ización. Una
Resumen
11
ez la ados los hid ogeles, se es udió su capacidad de einco po ación
de aciclo i o alaciclo i , po inme sión en disoluciones acuosas de
cada á maco. Se comp obó que los discos p esen aban mayo
a inidad po alaciclo i . A con inuación, se lle ó a cabo un es udio
de cesión con ambos á macos en FLS, lo que con i mó que los
hid ogeles imp esos y ca gados con alaciclo i p esen aban pe iles
de cesión más adecuados, con una libe ación sos enida du an e 10 h
que da luga a ni eles e apéu icos. Los hid ogeles se ca ac e iza on
en é minos de hinchamien o, ansmi ancia y p opiedades mecánicas,
ob eniéndose alo es simila es a los que son comunes pa a las LC
hid ó ilas. También se some ie on a un ensayo de i i ación en
memb ana co ioalan oidea de hue o (HET-CAM), no obse ándose
hemo agia, lisis ni coagulación. Finalmen e, con los hid ogeles con
mejo es p opiedades de ca ga y cesión de alaciclo i se lle a on a
cabo ensayos de pe meabilidad en có nea y escle a bo inas. De la
misma o ma que en el caso de la disolución acuosa de alaciclo i , el
á maco cedido po los hid ogeles ue capaz de acumula se en la
co nea y de pene a a a és de la escle a. Los esul ados ob enidos
indican que los hid ogeles p epa ados con MAA como monóme o
uncional e imp esos con alaciclo i son buenos candida os pa a la
adminis ación ópica ocula de es e á maco.
3) Diseño de hid ogeles bioinspi ados uncionalizados con
ci osina pa a la adminis ación ocula de ácido ans e úlico. El ácido
ans e úlico es uno de los an ioxidan es na u ales más po en es y
ÁNGELA VARELA GARCÍA
12
puede elimina especies eac i as de oxígeno (ROS) y de ni ógeno
(RNS), así como egula los sis emas ci op o ec o es. A ni el ocula ,
es ú il en el a amien o de lesiones co neales y puede sup imi la
p oducción de amiloide a ni el del c is alino. El p incipal
incon enien e es que su biodisponibilidad o al es in e io al 20%, po
lo que esul a con enien e diseña o mulaciones que pe mi an su
adminis ación ópica ocula . La c eación de ecep o es a i iciales
u ilizando la écnica de moldeado molecula (molecula imp in ing)
no es aplicable cuando la molécula de in e és es un an ioxidan e, ya
que se deg ada ía du an e la polime ización y, además, ha ía que és a
uese incomple a. Po lo an o, el desa ollo de hid ogeles con a inidad
po an ioxidan es equie e la iden i icación de g upos uncionales que
puedan ac ua como ecep o es, sin necesidad de lle a a cabo la
polime ización en p esencia de la molécula de in e és. Muchos
á macos basan su mecanismo de acción en su capacidad pa a
in e acciona con las bases pú icas (adenina y guanina) o pi imidínicas
( imina, ci osina y u acilo), que cons i uyen el ADN y el ARN. Es a
e apa de la Tesis se plan eó pa iendo de la hipó esis de que la
inco po ación de una base pi imidínica, como la ci osina, a la
es uc u a de un hid ogel debe do a lo de a inidad po las moléculas
con es uc u a complemen a ia en é minos de capacidad pa a
es ablece puen es de hid ogeno e in e acciones hid o óbicas п-п. La
u ilización de bases ni ogenadas como g upos uncionales no ha sido
ensayada p e iamen e, po lo que pa a su inco po ación a los
Resumen
13
hid ogeles se puso a pun o un p ocedimien o de anclaje pos -
polime ización.
Pa a lle a a cabo el es udio, los hid ogeles se p epa a on
mezclando HEMA con glicidilme ac ila o (GMA) y
e ilenglicol enile e me ac ila o (EGPEM) en dis in as p opo ciones.
El GMA se u ilizó como puen e pa a inmo iliza ci osina en los
hid ogeles, mien as que el EGPEM se inco po ó pa a e alua la
posibilidad de e o za la in e acción en e el ácido ans e úlico y la
ci osina o mando complejos de Rebek. Los hid ogeles se p epa a on
con un espeso de 0.45 mm y la uncionalización con ci osina se lle ó
a cabo po inme sión en una disolución de ci osina en agua:dioxano
(1:1 ol/ ol), a 80 °C du an e 24 h. T as el la ado, la p esencia de
ci osina se con i mó isualmen e bajo luz ul a iole a, pues o que es
una molécula al amen e luo escen e, y ambién median e
espec oscopía FTIR-ATR y análisis elemen al.
Los hid ogeles se ca ac e iza on en cuan o a g ado de
hinchamien o, ansmi ancia y p opiedades mecánicas, ob eniéndose
alo es si uados den o de los in e alos admi idos pa a LC
hid o ílicas.
La inco po ación de ácido ans e úlico se lle ó a cabo
sume giendo discos de hid ogel en una disolución acuosa del á maco.
Los hid ogeles uncionalizados con ci osina die on luga a una mayo
inco po ación de agen e an ioxidan e, has a 2.5 eces supe io y, po
lo an o, a coe icien es de epa o en amado/agua (KN/W) más

ÁNGELA VARELA GARCÍA
14
ele ados que los hid ogeles sin uncionaliza . Los ensayos de cesión
in i o, que se lle a on a cabo en FLS, e ela on que los hid ogeles
con mayo KN/W dan luga a pe iles de cesión más sos enidos. A
con inuación, los hid ogeles se some ie on a un ensayo de
compa ibilidad con memb ana co ioalan oidea de hue os ecundados
(ensayo HET-CAM), que es un mé odo al e na i o al ensayo in i o
en animales pa a e alua el iesgo de i i ación ocula . También se
e aluó la compa ibilidad de los hid ogeles con células epi eliales de la
có nea humana (HCEC) median e la p ueba WST-1. Ambos ensayos
con i ma on que los hid ogeles son al amen e biocompa ibles.
Los hid ogeles con mejo es p opiedades de ca ga y cesión de
ácido ans e úlico se ensaya on en cuan o a ac i idad an ioxidan e,
median e el es ORAC. Los esul ados ob enidos indica on que la
inco po ación del ácido ans e úlico a los hid ogeles no de e io a su
ac i idad an ioxidan e.
Finalmen e, se lle ó a cabo un es udio de pe meabilidad a a és
de có nea y escle a. Al igual que el ácido ans e úlico lib e en
disolución, el que se cedió a pa i de los hid ogeles mos ó una
ele ada endencia a acumula se en có nea y escle a, y ambién a pasa
al medio ecep o . Los esul ados ob enidos indican que la u ilización
de ci osina como componen e uncional ep esen a una nue a
es a egia pa a do a a los hid ogeles de a inidad po moléculas que,
como el ácido ans e úlico, cuen an en su es uc u a con g upos
Resumen
15
a omá icos y o os g upos con capacidad pa a o ma puen es de
hid ógeno.
En su conjun o, los esul ados de es a Tesis Doc o al ab en
nue as posibilidades pa a desa olla o mas de aplicación ópica
ocula , an o líquidas como sólidas, que p opo cionen ni eles
sos enidos de á maco en có nea y escle a.
In oduc ion
ÁNGELA VARELA GARCÍA
24
Figu e 1.4: Schema ic ep esen a ion o he laye s o he co nea and he cells ha
con o m i . Adap ed om Mas e on e al. (2018). This is an open access a icle
unde he CC BY-NC-ND license.
The u ea is loca ed be ween he e ina and he scle a, and is
o med by he cilia y body, he cho oid and he i is. I is a pigmen ed
ascula laye . The cilia y body co e s he an e io scle a. The cho oid
is he main pigmen ed ascula issue o he eyeball. The i is is loca ed
be ween he co nea and he lens, wi hin he aqueous humo , and
di ides he an e io and pos e io chambe s wi h a cen al ape u e, he
pupil (Malho a e al., 2011).
The c ys alline o lens is made up o long ib e cells, wi h
p o eins and wa e . I is di ided in o wo pa s, nucleus and co ex. I
is a anspa en and lexible issue, so ha ligh can pass h ough i
wi hou p oblems and ocus on he e ina. I can change he cu a u e

1. In oduc ion
25
o he su ace when necessa y, o adjus he ocal dis ance (Pe ash
2013; Wang e al., 2019).
The i eous humo ep esen s 80% o he olume o he eyeball
and is di ided in o h ee pa s: he i eous nucleus, he i eous base
and he i eous co ex. I is composed o 99% wa e , oge he wi h
ino ganic sal s, suga s, lipids and p o eins. I s unc ion, in addi ion o
p o iding mechanical suppo , is o main ain homeos asis in
neighbou ing issues, p o ide nu ien s, gua an ee op ical anspa ency
and p o ec agains he en y o cells o mac omolecules ha may
in e e e wi h he no mal unc ion o he eye (Mon ei o e al., 2015).
The e ina is a issue ha co e s he in e nal su ace o he
eyeball. I consis s o six classes o neu ons: pho o ecep o s (cones
and ods), bipola cells, ho izon al cells, amac ine cells, ganglion cells
and Mülle 's glia, a anged in se e al pa allel laye s. I s unc ion is o
con e signals coming om ou side in o ne e impulses, ansmi ed
om he op ic ne e o he b ain (Willoughby e al., 2010).
The op ic ne e is pa o he cen al ne ous sys em. I is made
up o e inal ganglion cell axons and suppo ing glial cells. I s unc ion
is o ansmi elec ical signals om he e ina o he b ain (Chen e
al., 2017).
The cho oid is a ascula laye loca ed a he back o he u ea and
consis s o i e laye s: B uch’s memb ane, he cho oiocapilla is, wo
ascula laye s (Halle ’s and Sa le ’s) and he sup acho oidea. I is
composed p incipally o blood essels. I s main unc ion is o supply
ÁNGELA VARELA GARCÍA
26
oxygen and nu ien s o he e ina, bu i can also ac as a
he mo egula o and modula o o in aocula p essu e (IOP) (Nickla
e al., 2010).
Finally, he scle a is a p o ec i e and suppo i e ou e laye o he
eye. I occupies he a ea be ween he co nea ma gin and he op ic
ne e, which cons i u es mo e han 80% o he eye's su ace. I s
s uc u e a ies wi h age, being hicke du ing he i s ew yea s o
li e. Then, i s e ches and becomes s i e . Scle a is o med by
collagen ib es ype I (90%) and ype III (5%), which a e included in
he p o eoglycan ma ix. I s unc ion is o main ain in aocula
p essu e and is whe e ex aocula muscles mee . In addi ion, i is he
main ca ie issue o he eye (Malho a e al., 2011; Coud illie e al.,
2015).
1.2. Eye-de ense mechanisms
Eye ana omy and physiology make i au onomous o de end i sel
om ex e nal agen s (Gaudana e al., 2010). The eye's de ense
mechanisms can be classi ied in o h ee ypes: mechanical, ana omical
and immunological (Akpek e al., 2003). The e a e many ac o s ha ,
in combina ion, p o ide comple e p o ec ion o he eye, o example,
he con inui y o co neal and scle al issue, blinking and ea
composi ion, among o he s (McClellan 1997). The p oblem a ises
when hese eye de ense mechanisms also ac as ba ie s o d ug
deli e y. The main ba ie s a e ca ego ized as lac imal ilm, co neal
and non-co neal ba ie s and blood ocula ba ie s (Figu e 1.5).
1. In oduc ion
27
Figu e 1.5: Schema ic ep esen a ion o he di e en de ense ba ie s ha he eye
p esen s o he adminis a ion o d ugs. Adap ed om Huang e al. (2018) wi h
pe mission om Else ie .
1.2.1. Lac imal ilm
I is he i s p o ec i e ba ie o he eye. I consis s o p o eins,
lipids and mucin, wi h pe ec elec oly e composi ion and op imal
nu ien and pH alues. The lac imal luid is made up o a lipid laye ,
an aqueous laye and a mucous memb ane, om he ou side o he
inside. I s unc ion is o modula e cell mig a ion and p oli e a ion
du ing healing, as well as o modula e no mal cell di e en ia ion and
ÁNGELA VARELA GARCÍA
28
he sec e ion o elec oly es and wa e . The low o ea s is cons an ,
being enewed e e y 2-20 minu es, so he esidence ime o any d ug
on he ocula su ace is e y low, which educes i s possibili y o
abso p ion (Ba a e al., 2009; Huang e al., 2018).
1.2.2. Co neal ba ie
The co nea, wi h a hickness o 0.5 mm and o med mainly by
collagen, is he main ba ie o he pene a ion o molecules in o he
eye (Sánchez-López e al., 2017; Huang e al., 2018). I consis s o
i e laye s, each o which has di e en p ope ies. The ou e mos
laye is he epi helium (s a i ied, squamous and no ke a inized),
which may allow he pass o small hyd ophobic molecules bu hinde s
he en ance o hyd ophilic d ugs. Only molecules wi h a molecula
weigh below 500 Da can pass h ough he pa acellula pa hway
(Ba a e al., 2009; Huang e al., 2018). The s oma (in e media e
laye ) opposes o he passage o lipophilic molecules due o i s high
con en in hyd a ed collagen and p o eoglycans. The endo helium is
he inne mos laye and consis s o a monolaye o polygonal cells.
Like he epi helium, i p e en s he passage o hyd ophilic molecules
o he aqueous humo bu allows he passage o small lipophilic
molecules (Ba a e al., 2008; Huang e al., 2018). In he co nea,
collagen ibe s a e p esen in he Bowman's laye and s oma. In he
i s one, he ib ils ha e a diame e be ween 20-25 nm and hei
dis ibu ion is andom, o ming lea es 8-12 µm hick. In he s oma,
he diame e o collagen ibe s a ies be ween 25-35 nm and i s
1. In oduc ion
29
dis ibu ion is in pa allel, o ming la lamella bundles (Komai e al.,
1991). The physicochemical p ope ies o d ugs also a ec hei
pene a ion h ough he co nea. Adequa e balance be ween lipophilia,
molecula weigh and deg ee o ioniza ion is equi ed o a success ul
passi e di usion. The mos common way o passage o d ugs h ough
he co nea is anscellula , while he pa acellula p edomina es o
hyd ophilic o low molecula weigh d ugs (Ba a e al., 2009;
Sánchez-López e al., 2017).
1.2.3. Non-co neal ba ie s
The conjunc i a is a hin mucous memb ane ha co e s he
eyelids in e nally and he an e io su ace o he scle a. Being o med
by blood capilla ies and conjunc i al lympha ics, p oduc i e
abso p ion o a d ug is e y low, al hough he pe meabili y is g ea e
han in co nea and also allows he passage o hyd ophilic molecules
and molecula weigh s up o 10 kDa (Sánchez-López 2017; Huang e
al., 2018).
The scle a consis s mainly o collagen and mucopolysaccha ide.
Collagen ibe s a y in size om 25 o 230 nm. Al hough hey o m
lamella bundles, indi idual ib ils a e mo e andomly a anged han
in he co nea. Thickness a ies om 0.5-6 µm. In he ou e mos pa
o he scle a, he collagen bundles a e na owe and hinne han in he
inne pa (Komai e al., 1991). Scle a has a su ace a ea o abou 16
cm2 and i is mo e pe meable o hyd ophilic solu es han o he ocula
s uc u es, such as he co nea, as hey can di use h ough he aqueous

ÁNGELA VARELA GARCÍA
30
medium be ween collagen ib ils, a he han h ough cell memb anes.
In his case, he adius o he molecules be e p edic s pe meabili y
han molecula weigh (MW). Thus, molecules wi h highe MW bu
smalle adius c oss he scle a be e han o he s o he same MW bu
la ge adius. Molecula cha ge also in luences he anspo o
molecules; nega i ely cha ged molecules a e mo e pe meable han
hose wi h posi i e cha ge, due o he cha ge o he p o eoglycan
ma ix, which is nega i e. O e all, d ug pe meabili y h ough scle a is
a o ed compa ed o co nea (Ba a e al., 2008; Huang e al., 2018).
1.2.4. Blood ocula ba ie s
The e exis wo ypes o ocula ba ie s in cha ge o egula ing he
solu es ha c oss owa ds he in e nal zones o he ocula globe: he
haema o-aqueous ba ie (BAB) and he haema o- e inal ba ie
(BRB).
The BAB is loca ed a he an e io egion and consis s o he
endo helium o he i is/cilia y blood essels and he non-pigmen ed
cilia y epi helium. I s unc ion is o egula e he in aocula p essu e,
u ning he low o aqueous humo , o main ain he anspa ency and
chemical composi ion o he ocula luids. I also egula es he passage
o d ugs om he an e io o he pos e io segmen . The BRB is
loca ed a he pos e io egion o he eye and also consis s o wo ypes
o cells: capilla y endo helial cells o he e ina and cells o he e inal
pigmen epi helium, which o m he in e nal and ex e nal BRB,
espec i ely. I s main unc ion is o hinde he di usion o subs ances
1. In oduc ion
31
om he ci cula o y o en o he e ina. The main limi ing p ope y
o he passage o subs ances is he molecula adius. Thus, he
pe meabili y dec eases as he adius inc eases. Lipophilia also
in luences; he e o e, only small and lipophilic molecules can pass
om he cho oid o he e ina (Huang e al., 2018).
1.3. Ocula d ug adminis a ion
D ug adminis a ion a he an e io segmen o he eye can be
done in se e al o ms. Topical adminis a ion is he mos common
ou e o he ea men o diseases o he an e io segmen , usually as
eye d ops o oin men s. The ollowing ad an ages can be men ioned:
i is a non-in asi e and painless pa hway wi h high pa ien
compliance; e y high doses o he d ug a e no needed; and he e ec
may be immedia e. The main disad an age is ha ocula
bioa ailabili y o d ugs adminis e ed opically is less han 5% due o
bo h physicochemical ba ie s ( he s uc u e o he eye o he
composi ion o he ea ) as well as naso-lac imal d ainage, ea ing o
blinking e lexes o he low olume ha can hos he cul-de-sac. In
addi ion, i is es ima ed ha he ea olume is 7 μL, and he
es o a ion ime o he ea ilm is as (2-3 minu es), so ha opically
adminis e ed solu ions would be elimina ed sho ly a e ins illa ion
(Awwad e al., 2017; Djebli e al., 2017). Mo eo e , sys emic side
e ec s may a ise, as much o he ins illed dose passes in o he
bloods eam h ough he conjunc i a and nasal mucosa. In addi ion,
nasolac imal d ainage o ce ain subs ances can cause oxic eac ions
ÁNGELA VARELA GARCÍA
32
(Lo sson e al., 1999; Ribei o e al., 2015). This sys em connec s he
low o ea s om he eye o he nasal ca i y. The pa o he d ug
d ained a e opical applica ion passes in o he lac imal sac and hen
in o he nasolac imal duc un il i eaches he nose. Du ing his
passage, he d ug passes h ough ascula ized a eas, whe e i can be
abso bed in o he sys emic ci cula ion, which can lead o undesi able
side e ec s (Bachu e al., 2018).
Oph halmic p epa a ions mus mee some speci ic equi emen s.
The ac i e molecule mus ha e a ce ain aqueous solubili y. In
addi ion, he e a e se e al c i ical pa ame e s ha need o be
moni o ed. The oph halmic o mula ions mus ha e a ole able acidi y,
wi h an adequa e pH a ound 7.4, al hough he e a e some excep ions.
They mus also be iso onic as well as s able a oom empe a u e.
O he c i ical ac o s o conside a e d ug pKa and o mula ion
iscosi y. Excipien s used du ing manu ac u e mus be ee om
oxici y and should no in e ac wi h he packaging. All oph halmic
p oduc s mus be s e ile, and injec ables mus also be ee o
endo oxins and pa icles (No ack e al., 2016; Yellepeddi e al., 2016)
(Figu e 1.6). D ugs can be adminis e ed o he an e io segmen o he
eye also hough in acame al and subconjunc i al injec ions. Bo h
a oid he co nea and hema oencephalic ba ie and p o ide high
ocula bioa ailabili ies. Ocula injec ions p esen all he
incon eniences associa ed o injec able o mula ions, agg a a ed by
he sensi i e egion whe e he o mula ions a e deli e ed, which
1. In oduc ion
33
causes pa ien discom o and has he isk o issue damage and
in ec ion. Finally, he sys emic o al ou e despi e being a non-in asi e
is ba ely used, since e y high doses o d ug a e necessa y o exe an
e ec a he ocula le el and he e a e many ad e se e ec s on o he
issues (Janagam e al., 2017).
Figu e 1.6. Pene a ion and elimina ion pa hways o d ug a e opical
adminis a ion. Adap ed om Janagam e al. (2017) wi h pe mission om Else ie .
The adminis a ion o d ugs o he pos e io segmen o he eye is
mo e complica ed. Topical o ms, such as d ops o oin men s, can
ha dly each he pos e io segmen , excep i he d ug can e icien ly
en e h ough he conjunc i a-scle a pa hway. D ugs can be
adminis e ed o ally, in amuscula ly o in a enously, bu he as
majo i y o d ugs canno c oss he ba ie s, so bioa ailabili y is
ÁNGELA VARELA GARCÍA
40
wo ldwide, and his igu e is expec ed o double in 30 yea s (Tan e
al., 2019).
Reac i e oxygen species (ROS) a e a subp oduc o no mal
anae obic me abolism. Enzymes such as SOD (supe oxide dismu ase),
CAT (ca alase) o GPx (glu a hione pe oxidase) neu alize hese
subp oduc s. When a disequilib ium is gene a ed in he edox
haemos asis o he p o- and an ioxidan sys ems due o he incapaci y
o enzymes o elimina e ee adicals, cell nec osis is igge ed due o
damage o p o eins, lipids and DNA, causing degene a ion a he
ocula le el (Bungau e al., 2019; Tan e al., 2019).
The e a e di e en ac o s ha p omo e oxida i e s ess, such as
age, exposu e o ligh o me abolic p ocesses, such as hype glycemia
(Bungau e al., 2019). The co nea p o ec s he eye om en i onmen al
s ess by abso bing ul a iole (UV) ligh , bu p olonged i adia ion
may cause co neal lesions (Ze nii e al., 2018). The adia ion can end
up damaging he an e io segmen . The mos common acu e condi ion
is pho oke a i is and in he long- e m ca a ac s, ca cinomas,
melanomas o o he pa hologies o he conjunc i a may appea . The
mos se ious consequence o UV adia ion is ROS gene a ion. A he
ocula le el, he e a e se e al an ioxidan s o low molecula weigh in
bo h issues and luids, such as asco bic acid, alpha- ocophe ol o
glu a hione, and o high molecula weigh , which a e he enzymes
men ioned abo e. The co nea, especially he an e io pa , can abso b
up o 60% o UVA adia ion and up o 92% o UVB adia ion,

1. In oduc ion
41
al hough i is mo e sensi i e o he la e . Al hough he eye has an
an ioxidan de ense, when he e is an inc ease in UV adia ion, he e is
a p ooxidan /an ioxidan imbalance ha a o s he damage (Cejko a e
al., 2004).
Se e al ca o enoids and polyphenols ha e an ioxidan and an i-
in lamma o y ac i i ies. They dec ease he p oduc ion o ROS, which
in u n inhibi s umo nec osis ac o α (TNFα) and ascula
endo helial g ow h ac o pa hways, which helps o supp ess p53-
dependen apop osis, elimina ing he genesis o in lamma o y ma ke s
(IL-8, 6, 1a and endo helial leucocy e adhesion molecule-1) (Bungau
e al., 2019). One example is es e a ol, which has also been shown
o exhibi an i-aging p ope ies a ocula le el (Abu-Ame o e al.,
2016). Ano he compound o highligh is ans e ulic acid, which
besides being a powe ul an ioxidan , exe s an i-in lamma o y and
an ibac e ial e ec s, among o he s. I s mechanism o ac ion, in
addi ion o elimina ing ee adicals, can inhibi he enzymes ha
ca alyze he syn hesis o hese ee adicals (Zdunska e al., 2018). A
he ocula le el, s udies ha e shown ha i can be use ul o he
healing o co neal wounds (Tsai e al., 2016; G imaudo e al., 2020),
o o supp ess he p oduc ion o amyloid B in he human lens (Nagai
e al., 2017).
1.5. Ocula i al in ec ions
I is es ima ed ha 20-70% o conjunc i i is a e i al, and o
hese, 65-90% a e caused by adeno i uses. These i al in ec ions
ÁNGELA VARELA GARCÍA
42
sp ead h ough he ai , by deposi s, o by di ec con ac wi h he i us.
Conjunc i i is can be classi ied in wo g oups: papilla y o ollicula .
The i s one mani es s i sel wi h la and compac p ojec ions, wi h
nume ous eosinophils, lymphocy es, plasma cells and mas cells in he
s oma ha su ounds a cen al ascula channel. I is usually
associa ed wi h a o eign body esponse o an alle gic immune
esponse. Follicula conjunc i i is occu s wi h p ominen ollicles in
he lowe palpeb al and o niceal conjunc i a. The mos common
symp oms and signs du ing i al conjunc i i is a e o eign body
sensa ion, ed eyes, i ching, sensi i i y o ligh , bu ning, and wa e y
discha ge (Li e al., 2018; Solano e al., 2019).
Ke a i is is ano he eye in ec ion caused by a i us; in his case
mainly he pes simplex i us (HSV). The numbe o people a ec ed by
his disease wo ldwide was es ima ed a 1.5 million. I is he mos
common cause o unila e al in ec ious co neal blindness. Unlike
bac e ial o ungal ke a i is, i al ke a i is can be ecu en and
ch onic. The e a e wo o he o ms o i al ke a i is, bu hey a e less
common: a icella-zos e i us (VZV) ke a i is and cy omegalo i us
(CMV) ke a i is (Aus in e al., 2017). The classic symp oms o i al
ke a i is a e eye pain, blu ed ision, edness, and pho ophobia (Rowe
e al., 2013).
He pes zos e is a ele an eye in ec ion. I has i s o igin in he
eac i a ion o he a icella-zos e i us (VZV), a i us o he
he pes i idae amily (Table 1.1). Ini ially, he disease p esen s i sel
1. In oduc ion
43
as chickenpox, in ec ing he senso y ganglia. I usually occu s in he
ea ly s ages o li e. I is e y con agious bu benign and occu s in he
o m o blis e s sp ead h oughou he body wi h i ching.
Table 1.1. Classi ica ion o He pes i us.
He pes i idae amily
Sub- amily Types Vi us
Alpha-he pes i inae
He pes i us 1 He pes simplex 1
He pes i us 2 He pes simplex 2
He pes i us 3 Va icella Zos e
Be a-he pes i inae
He pes i us 5 Cy omegalo i us
He pes i us 6 He pes i us lympho ope
He pes i us 7 Human he pes i us
Gamma-he pes i inae He pes i us 4 Eps ein-Ba Vi us
He pes i us 8 Kaposi's Sa coma
The eac i a ion o he i us, known as he pes zos e o shingles,
is mo e dange ous. I mani es s as esicula e up ions, a ec ing
mos ly he ho acic ne e, bu also he igeminal ne e. Fac o s such
as changes in T-cells o a dec ease in he neu aliza ion o an ibodies,
which occu as age p og esses, in luence he possible eac i a ion o
VZV. The isk is also highe in immunocomp omised pa ien s. The
da a wa n ha abou 50% o adul s a ec ed by he pes zos e a e a isk
o complica ions. He pes zos e oph halmicus ep esen s be ween 10-
20% o cases o zos e and is he second mos common complica ion.
The disease begins wi h symp oms o se e e pain and discom o .
ÁNGELA VARELA GARCÍA
44
A e a ew days, skin lesions appea . Complica ions a he le el o he
co nea appea in 65% o cases. The acu e phase p esen s wi h
epi helial ke a i is (punc u e o dend i ic) o s omal ke a i is, and he
la e phase wi h neu o ophic ke a opa hy o neo ascula iza ion o he
co nea. The mos common mani es a ions a e pho osensi i i y,
dec eased ision o pe o a ion, among o he s (Zhu e al., 2014).
1.5.1. T ea men o ocula i al in ec ions
The causal agen o i al conjunc i i is should be iden i ied
co ec ly so ha p ope ea men can be gi en. Fo example,
commonly i al conjunc i i is occu s wi h wa e y discha ge, as
opposed o bac e ial conjunc i i is ha p esen s mucopu ulen
discha ges. The e is no e ec i e ea men in i s en i e y. A i icial
ea s, opical an ihis amines, o cold comp esses a e used as
pallia i es. The e ec i eness o ocula an i i als is no high. Topical
an ibio ics a e no indica ed since hey a e no capable o p e en ing
seconda y in ec ions, also causing undesi ed side e ec s, such as
alle gy o oxici y. O all cases o acu e conjunc i i is, he he pes
simplex i us is esponsible o 1.3-4.8%. This ype o pa hology is
usually unila e al, wi h wa e y discha ge and some imes e en
esicula lesions may appea on he eyelid. The usual ea men
consis s o opical and o al an i i als. The use o opical
co icos e oids should be a oided because hey a e po en ia o s o he
i us (Aza i e al., 2013).
1. In oduc ion
45
On he o he hand, opical ea men o i al ke a i is caused by
he pes simplex i us (HSV) includes an i i al d ugs and adjunc i e
opical co icos e oids. The mos commonly used opical an i i al was
i lu idine, al hough i s bioa ailabili y was e y low and caused
su ace oxici y a he ocula le el, so i s use has been educed.
Acyclo i is he i s choice o he ea men o ke a i is since i s
e ec i eness is high and i is less oxic. New syn he ic d ugs, such as
ganciclo i , ha e a b oade -spec um an i i al ac ion, i.e., in addi ion
o ea ing ke a i is caused by HSV, i also a acks VZV and CMV.
Addi ionally, opical co icos e oids a e some imes used as adju an
he apy. On he o he hand, acyclo i is also gi en as an o al adju an
he apy. Valacyclo i was also seen o ha e g ea e o al
bioa ailabili y o he ea men o HSV, equi ing lowe doses (Aus in
e al., 2017).
Finally, he pes zos e oph halmicus usually appea s as a mild
case, bu an i i al use is s ill ecommended o he i s 72 hou s a e
he onse o he ash. Ea ly ea men can lead o a educ ion in he
du a ion o he illness as well as acu e pain and hal e he likelihood o
eye diso de s. The d ugs chosen a e acyclo i , alacyclo i and
amcyclo i . Al hough all h ee ha e simila e icacies, he mo e
ecen ones ( alacyclo i and amcyclo i ) ha e highe o al
bioa ailabili y. In his case, co icos e oids a e also used as adju an
he apy, especially du ing he i s s age o he disease, o elie e pain
and imp o e healing, bu hei long- e m use should be a oided

ÁNGELA VARELA GARCÍA
46
because o side e ec s. I he disease mani es s la e, an i i al d ugs a e
no e ec i e and opical co icos e oids should be used o educe
in lamma ion (Zhu e al., 2014).
1.6. Micelles and con ac lenses as pla o ms o ocula deli e y
The classic me hods o ocula adminis a ion o d ugs (eye d ops
o oin men s) ha e di e en d awbacks, as explained in sec ion 1.3.
Tha is he eason o a con inuous sea ch o new d ug dosage o ms,
o imp o e d ug ocula bioa ailabili y, combining an e icien
con olled elease and minimizing pain and discom o o he pa ien .
Among o he pla o ms, polyme ic micelles and so con ac lenses a e
gaining inc easing in e es (Go e e al., 2019).
1.6.1. Polyme ic micelles
The use o nanoca ie s is gaining inc easing a en ion o he
deli e y o ocula d ugs (Gomez-Balles e os e al., 2019). Polyme ic
micelles a e o med by amphiphilic copolyme s, which spon aneously
sel -assemble in an aqueous medium, once hey exceed he c i ical
micella concen a ion (CMC) (G imaudo e al., 2019). Thei size can
a y be ween 5-100 nm, and hei shapes can also be di e en . Du ing
he p ocess o micelle o ma ion he e is an equilib ium o
in e molecula o ces, such as an de Waals o ces, hyd ogen
bonding and hyd ophobic, s e ic and elec os a ic in e ac ions
(Gauche e al., 2005). The s uc u e o he micelle comp ises an
in e nal hyd ophobic co e, capable o hos ing liposoluble d ugs and
solubilizing hem, and an ex e nal hyd ophilic shell, in con ac wi h
1. In oduc ion
47
he ex e nal en i onmen , which physically s abilizes he micelle
(Figu e 1.7) (C oy e al., 2006).
Figu e 1.7. Schema ic ep esen a ion o he s uc u e o a polyme ic micelle. Figu e
made by he au ho o his Thesis.
The encapsula ion o d ugs in polyme ic micelles p e en s he
in e ac ion wi h he su ounding en i onmen , which enhances hei
physicochemical s abili y. In addi ion, p ope ies o he ex e nal shell,
such as iscosi y, hickness o po osi y, may de e mine he a e o
d ug elease. The p ocess o d ug inco po a ion is complex and
depends on he molecula and physicochemical p ope ies o bo h
pa s (micelle and d ug). The molecula weigh and he hyd ophilic-
lipophilic balance (HLB) a e p ope ies o conside in he copolyme ;
o simila molecula weigh s, a dec ease in HLB leads o la ge
nucleus and wi h g ea e encapsula ion powe . On he o he hand, o
simila HLB, he highe he molecula weigh o he copolyme , he
mo e e icien he encapsula ion is. P ope ies o he d ug such as
molecula weigh , adius, lipophilia (pa i ion coe icien , logP),
ÁNGELA VARELA GARCÍA
48
mel ing poin , endency o agg ega ion and he p esence o speci ic
unc ional g oups ha may in e ac wi h he micelle modi y he
encapsula ion e iciency.
The e a e di e en me hods o he p epa a ion o d ug-loaded
polyme ic micelles. The mos di ec and simple me hod consis s o
dissol ing he copolyme in wa e , s abilizing he micelle dispe sion a
a sui able empe a u e (48-72 h) and hen adding he d ug o be
inco po a ed in o he micelles. This me hod is gene ally used o
micelles o med by copolyme s o in e media e HLB. I may equi e
he applica ion o hea o dehyd a e he segmen s ha will o m he
nucleus o o m he micelles. Ano he echnique, used o mo e
hyd ophobic copolyme s, consis s o dissol ing bo h componen s
(copolyme and d ug) in wa e -miscible o ganic sol en s. The
mechanism o micella o ma ion will depend on he p ocedu e by
which he o ganic sol en is emo ed. Fo example, he mix u e may
be dialyzed and he slow elimina ion o he o ganic phase igge s he
o ma ion o micelles; o he o ganic phase may e apo a e, o ming a
polyme ic ilm, which will be ehyd a ed wi h an aqueous sol en ,
aided by hea , o o m he d ug-loaded micelles. The e a e mo e
echniques, such as apping a hyd ophobic d ug in an O/W emulsion,
cas ing in solu ion o lyophiliza ion (Gauche e al., 2005).
Topical o mula ions o d ug-loaded polyme ic micelles can be
conside ed as non-in asi e deli e y sys ems. These sys ems can s ay
in he si e o adminis a ion long enough o he d ug o exe i s
1. In oduc ion
49
he apeu ic e ec due o p ope ies such as iscosi y o mucoadhesion.
In gene al, he con ac ime o he o mula ion is di ec ly p opo ional
o i s iscosi y. The clea ance o d ugs by blinking o naso-lac imal
d ainage is educed by inc easing he iscosi y o he sys em. The use
o mucoadhesi e componen s o he o ma ion o he polyme
micelles also inc eases he esidence ime o he micelles a he
adminis a ion si e due o he o ma ion o co alen bonds wi h mucin.
Polyme ic micelles a e sa e and e ec i e sys ems and a oid pa ien
discom o . Fo he d ug o each he pos e io segmen o he eye, i
can ollow he co neal o conjunc i al-scle al ou e (Figu e 1.8)
(Mandal e al., 2017; G imaudo e al., 2019).
The size o he polyme ic micelles in he nanoscale endows hem
wi h he abili y o pass h ough s uc u es such as he co nea o he
conjunc i a/scle a pa hway. The pass o hyd ophobic d ugs h ough
he hyd ophilic s oma may be a o ed by he encapsula ion in
nanomicelles. The scle a has a la ge a ea, which allows he polyme ic
micelles o sp ead la e ally o he pos e io segmen . F om he e, he
cells o he e inal pigmen ed epi helium may engul nanoca ie s by
endocy osis, opening he possibili y o exe ing an e ec on he ocula
issues o he pos e io segmen (Hughes e al., 2005; Mandal e al.,
2017).
ÁNGELA VARELA GARCÍA
56
a e. C i ical pa ame e s such as he selec ion o he unc ional
monome and i s s oichiome y wi h espec o he empla e
mus be conside ed (Al a ez-Lo enzo e al., 2010).
g) Supe c i ical luid imp egna ion: Supe c i ical luids, mainly
supe c i ical CO2 (scCO2) can be used o enhanced d ug
dissolu ion and pene a ion in o polyme ne wo ks (Ga cia-
Gonzalez e al., 2015). The p ocess s a s wi h he dissolu ion
o he d ug in he supe c i ical sol en in con ac wi h he CLs.
This echnique allows he loading o hyd ophilic and
hyd ophobic d ugs a highe amoun han con en ional
soaking in aqueous medium, bu no signi ican imp o emen s
in he con ol o elease a e a e usually achie ed (Yañez e al.,
2011).
h) Inco po a ion o colloidal nanopa icles: he adminis a ion o
d ugs encapsula ed in colloidal nanopa icles inc eases he
esidence ime o he d ug in he co nea and p e en s he
ocula enzymes om me abolizing he d ug. The e o e, he
addi ion o nanoca ie s o CLs may p olong he ac ion ime.
Ne e heless, he addi ion o nanopa icles may obs uc he
ision by dec easing he anspa ency o he lens.
The e a e ou me hods o p epa ing hese sys ems. The i s
one consis s o p epa ing he nanopa icles loaded wi h d ug,
and hei subsequen dispe sion in p e-monome mix u es, o

1. In oduc ion
57
o m CLs. The second me hod is o add su ac an s and d ugs
o p e-monome mix u es o o m micelles du ing
polyme iza ion. The hi d me hod consis s o imme sing he
al eady o med CL in a suspension o nanopa icles. Finally,
he ou h me hod consis s o immobilizing he nanopa icles
on he su ace o he CL by means o chemical bonds.
Di e en ypes o nanopa icles can be subjec ed o hese
p ocedu es, such as polyme nanopa icles, micelles,
liposomes o mic oemulsions (Mo ison e al., 2014; Ali e
al., 2016; Choi e al., 2018).
ÁNGELA VARELA GARCÍA
58
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2. Aims
73
2. Aims
The eye is a peculia o gan, p o ec ed by a combina ion o
ana omical, mechanical and immunological ba ie s. Al hough i is
easily accessible o opical d ug adminis a ion, he combina ion o
local and sys emic ba ie s p e en s a success ul d ug dis ibu ion.
Fo mula ions ha can combine he pa ien accep ance o opical
o mula ions and he e ec i eness o in aocula injec ions ha e been
la gely p ospec ed.
The aim o his PhD Thesis was o design polyme ic micelles and
CL wi h imp o ed ea u es o opical adminis a ion o oph halmic
d ugs. Speci ically, he esea ch ocused on he an i i al d ugs
acyclo i and alacyclo i and he an ioxidan agen ans e ulic acid.
These d ugs a e poo ly soluble in wa e and hei he apeu ic
ou comes could be no ably imp o ed i sus ained le els on he eye
s uc u es a e achie ed.
Acco ding o his gene al aim, h ee speci ic aims we e iden i ied
and de eloped as ollows.
1) Design o polyme ic micelles sui able o he adminis a ion o
acyclo i . This an i i al agen is he i s choice o he ea men o
ÁNGELA VARELA GARCÍA
74
ocula he pes caused by he he pes simplex i us. This i us can a ec
all laye s o he co nea, p oducing blu ed ision, edness, o ea ing.
He pes simplex ke a i is (in ec ion and in lamma ion o he co nea) is
he leading cause o in ec ion blindness wo ldwide. Acyclo i
pene a es in ec ed cells and compe es wi h na u al nucleosides o
inco po a e DNA and ac as a chain e mina o . A e ancho ing, a
suicidal inac i a ion mechanism occu s, since he e mina ed DNA
binds o he i al DNA polyme ase and i e e sibly inhibi s i ,
p e en ing i al eplica ion. Classic opical ea men equi es a long
applica ion pe iod and esul s in se ious side e ec s. O al
adminis a ion is cha ac e ized by ha ing a bioa ailabili y o less han
20%, which is he eason o p esc ibing high doses o long pe iods o
ime.
The hypo hesis o his i s s age o he Thesis was ha he
encapsula ion o acyclo i in polyme ic nanomicelles should inc ease
d ug solubili y o he d ug and p omo e he accumula ion o he d ug
in co nea and scle a and i s access o deepe s uc u es. Soluplus and
Solu ol copolyme s we e chosen as amphiphilic copolyme s. Soluplus
is a biodeg adable copolyme o poly inyl cap olac am-poly inyl
ace a e-polye hylene glycol (MW 90000-140000 g/mol, CMC 7.6
mg/L), which o ms aqueous dispe sions ha may unde go in si u
gelling a he ocula empe a u e. This addi ional ea u e may inc ease
he esidence ime a he si e o applica ion and enhance he con ol o
d ug elease. Solu ol o mac ogol 15-hyd oxys ea a e (MW 963.24
2. Aims
75
g/mol, CMC 0.005-0.02%) is a non-ionic su ac an , which imp o es
s abili y and solubili y o insoluble d ugs. I is s able, highly
biocompa ible and pe meable o he mucosa. To ca y ou he wo k,
dispe sions o each copolyme will be p epa ed co e ing a wide ange
o concen a ions and hei size, Z po en ial, capabili y o hos
acyclo i and heological beha iou will be e alua ed. Those
o mula ions combining adequa e pe o mances will be es ed
ega ding co nea and scle a accumula ion and pe meabili y.
2) Design o imp in ed hyd ogels o an i i al d ugs. Seeking o
p olonging d ug pe manence on he ocula su ace beyond ha he
o mula ions in nanomicelles may allow, he aim o he second s ep o
he Thesis was o design hyd ogels sui able o so CL wi h a ini y
o acyclo i and alacyclo i . Among he p oposed p ocedu es o
endow he hyd ogel CLs wi h 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 . This echnique equi es inco po a ing he
subs ances o in e es in o he monome s mix u e so ha hese 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 subs ance o in e es again. Func ional
monome s sui able o in e ac ion wi h he an i i al d ugs will be i s
sc eened using compu a ional modeling. Then hyd ogels will be
ÁNGELA VARELA GARCÍA
76
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 he d ug. The
load and elease capaci ies o an i i al d ugs will be e alua ed, as well
as hei biocompa ibili y in cho ioallan oic memb ane (HET-CAM
es ). The hyd ogels will be cha ac e ized in e ms o swelling,
ansmi ance and mechanical p ope ies. Finally, he pe meabili y
h ough bo ine co nea and scle a o he aqueous solu ion o he d ug
and he d ug eleased by he hyd ogels will be compa ed.
3) Design o cy osine- unc ionalized bioinspi ed hyd ogels o
ocula adminis a ion o ans e ulic acid. T ans e ulic acid is one o
he mos powe ul na u al an ioxidan s, i sca enges eac i e oxygen
and ni ogen species (ROS and RNS), and i egula es cy op o ec i e
sys ems. A he ocula le el, i is use ul in he ea men o co neal
lesions and can sup ess he p oduc ion o amyloid a he c ys alline.
Howe e ocula o mula ions o sus ained elease o ans e ulic acid
ha e no been de eloped ye . The c ea ion o a i icial ecep o s using
he molecula imp in ing echnique is no applicable when he
molecule o in e es is an an ioxidan since i would deg ade du ing
polyme iza ion and would also in e e e wi h he p ocess. The e o e,
he de elopmen o hyd ogels wi h an a ini y o an ioxidan s
equi es he iden i ica ion o unc ional g oups ha can ac as
ecep o s wi hou he need o ca ying ou he polyme iza ion in he
p esence o he molecule o in e es . The mechanism o ac ion o
2. Aims
77
se e al d ugs elies on hei abili y o in e ac wi h pu ic bases
(adenine and guanine) o py imidine bases ( hymine, cy osine and
u acil) ha build up DNA and RNA. The hypo hesis o he las pa o
he Thesis was ha he inco po a ion o a py imidine base, such as
cy osine, in o he s uc u e o a hyd ogel should endow i wi h an
a ini y o molecules wi h complemen a y s uc u e in e ms o abili y
o in e ac h ough hyd ogen bonding and hyd ophobic п-п s acking.
The use o ni ogenous base as unc ional g oups has no been
p e iously es ed, so a pos -polyme iza ion ancho ing p ocedu e was
de eloped o hei inco po a ion in o he hyd ogels. To ca y ou he
s udy, he hyd ogels will be p epa ed by mixing HEMA wi h di e en
p opo ions o glycidylme hac yla e (GMA) and e hylene
glycolphenyl me hac yla e (EGPEM). GMA can se e as a b idge o
immobilize cy osine in hyd ogels, while EGPEM may ein o ce he
in e ac ion be ween ans e ulic acid and cy osine o ming Rebek
molecula complexes. The hyd ogels will be unc ionalized wi h
cy osine and cha ac e ized ega ding he amoun g a ed and in e ms
o deg ee o swelling, ansmi ance and mechanical p ope ies. The
capabili y o he hyd ogels o load ans e ulic acid and o sus ain i s
elease while p ese ing he an ioxidan ac i i y will be e alua ed in
de ail. HET-CAM es and iabili y o human co neal epi helial cells
(HCEC) will be used o a i s assessmen o he ocula compa ibili y
o he de eloped hyd ogels. Finally, pe meabili y o ans e ulic acid

ÁNGELA VARELA GARCÍA
78
h ough co nea and scle a when applied in solu ion and when
deli e ed om he hyd ogels will be compa ed.
Polyme ic micelles o acyclo i ocula deli e y:
o mula ion and co nea and scle a pe meabili y
3. Polyme ic micelles o acyclo i ocula deli e y: o mula ions and co nea
and scle a pe meabili y
81
3. Polyme ic micelles o acyclo i ocula
deli e y: o mula ion and co nea and scle a
pe meabili y
3.1. In oduc ion
The eye is conside ed a unique issue om an immunological
pe spec i e. Se e al p o ec i e elemen s a e p esen on he ocula
su ace; o example, mucins ha o m a dense glycocalix on he
co nea p o ide a physical ba ie ha p e en s bac e ial adhesion, and
β-de ensins, calp o ec in, lysozyme, lipocalin and lac o e in exhibi
an imic obial ea u es. Eye su ace in ec ions appea when he
homeos asis is dis u bed by bo h un a o able en i onmen al
condi ions and in ec ious agen s, which b eak down he eye su ace
and al e he inna e immune sys em o his o gan (Caspi, 2013;
Pea lman e al., 2013; Lu e al., 2016). Ocula in ec ions can be
caused by bac e ia and ungi pa hogens (Caspi, 2013; Mangoni e al.,
2016) o by i uses such as He pes simplex, Va icella Zos e o
Cy omegalo i us (Edwa ds e al., 2017).
The a icella zos e i us (VZV) causes chickenpox and he pes
zos e . The i s is a benign disease and he second appea s a e a
ÁNGELA VARELA GARCÍA
88
1000N (TA Ins umen s, UK) heome e i ed wi h a Pel ie pla e and
a 6-cm in diame e cone (2 º). The angula equency was ixed a 5
ad/s and he oscilla ion s ess a 0.1 Pa.
3.2.4. Acyclo i solubiliza ion
Soluplus and Solu ol solu ions (3 mL) we e pou ed in 5 mL
Eppendo ubes con aining acyclo i in excess (50 mg). Solubili y o
acyclo i in wa e and PBS pH 7.4 was also eco ded. The sys ems
we e main ained unde magne ic s i ing o 72 h, a 300 pm and
oom empe a u e. Then, hey we e cen i uged a 5000 pm o 30
min o sepa a e non-solubilized acyclo i . Abso bance o he
supe na an s was measu ed a 252 nm (UV/Vis spec opho ome e
Agilen 8453, Ge many) p e ious dilu ion wi h wa e :e hanol 50:50
/ mix u es. Acyclo i concen a ion was calcula ed using a
calib a ion cu e p e iously p epa ed.
The solubili y da a was used o calcula e he ollowing pa ame e s
(Al a ez-Ri e a e al., 2016).
Mola solubiliza ion capaci y (moles o d ug ha can be solubilized
pe mol o copolyme o ming micelles):
𝜒=  
 [Eq. 3.1]
Micelle-wa e pa i ion coe icien ( a io be ween he d ug
concen a ion in he micelle and he aqueous phase):
𝑃 = 
 [Eq. 3.2]

3. Polyme ic micelles o acyclo i ocula deli e y: o mula ions and co nea
and scle a pe meabili y
89
Mola micelle-wa e pa i ion coe icien (which elimina es he P
dependence on he copolyme concen a ion, assigning a de aul
concen a ion o 1M):
𝑃𝑀 =∗( )
 [Eq. 3.3]
Gibbs s anda d- ee ene gy o solubiliza ion was es ima ed om he
mola micelle/wa e pa i ion coe icien (PM) and he micelle-wa e
pa i ion coe icien (P), as ollows
𝛥𝐺𝑠 = −𝑅𝑇∗ln(𝑃𝑀) [Eq. 3.4]
𝛥𝐺𝑠 = −𝑅𝑇∗ln(𝑃) [Eq. 3.5]
In hese equa ions, S o ep esen s he o al solubili y o acyclo i
in he micella solu ion, Sw is he acyclo i solubili y in wa e , CMC is
he c i ical micelle concen a ion, Ccopol is he copolyme
concen a ion in each micelle solu ion, and R is he uni e sal cons an
o gases.
3.2.5. Micelle s abili y agains dilu ion
Aliquo s o acyclo i -loaded Soluplus (12 %w/w) micelle
dispe sions p epa ed in PBS pH 7.4 we e pou ed on o empe a u e-
con olled (35 ºC) qua z cells al eady con aining PBS, so he
dispe sions we e dilu ed 30- o 60- old. The abso bance o he
samples was immedia ely measu ed a 252 nm and each 30 seconds
du ing 30 min (UV/Vis spec opho ome e Agilen 8453, Ge many).
Simila expe imen s we e also ca ied ou in iplica e using wa e and
SLF as dilu ion medium.
ÁNGELA VARELA GARCÍA
90
3.2.6. Co neal and scle a pe meabili y assay
Acyclo i pe meabili y assays h ough co nea and scle a we e
ca ied ou ollowing he BCOP es p o ocol (Al a ez-Ri e a e al.,
2016; OECD, 2017; Al a ez-Ri e a e al., 2018). Bo ine eyes we e
collec ed in he i s hou a e dead, om a local slaugh e house.
They we e anspo ed comple ely imme sed in PBS solu ion wi h
an ibio ics added (penicillin 100 IU/mL and s ep omycin 100 μg/mL)
and main ained in an ice ba h. Nex , co neas we e isola ed wi h 2-3
mm o su ounding scle a, o scle as we e isola ed. In bo h cases,
issues we e washed wi h PBS, be o e moun ed on e ical di usion
F anz cells. Dono and ecep o chambe s we e illed wi h ca bona e
bu e pH 7.2. The ecep o chambe s we e placed inside a ba h a 37
ºC and kep unde magne ic s i ing du ing 1 h in o de o balance
ocula issues. Then, he olume o he dono chambe was comple ely
emo ed and eplaced by d ug solu ions (2 mL). Acyclo i aqueous
solu ion (0.3 mg/mL) was p epa ed dissol ing he equi ed amoun in
wa e . Sa u a ed acyclo i solu ion in Soluplus micelles (20% w/w)
was p epa ed as epo ed abo e; he inal d ug concen a ion was 1.34
mg/mL. The chambe s we e co e ed wi h pa a ilm o p e en
e apo a ion (0.785 cm2 a ea a ailable o pe mea ion). Samples (1
mL) we e emo ed om he ecep o chambe a 0.5, 1, 2, 3, 4, 5 and
6 h, eplacing he same olume wi h ca bona e bu e each ime, and
aking ca e o emo ing bubbles om he di usion cells. All
expe imen s we e ca ied ou in iplica e.
3. Polyme ic micelles o acyclo i ocula deli e y: o mula ions and co nea
and scle a pe meabili y
91
Acyclo i pe mea ed was quan i ied by HPLC (Wa e s 717
Au osample , Wa e s 600 Con olle , 996 Pho odiode A ay De ec o ),
i ed wi h a C18 column (Wa e s Symme y C18, 5 μm, 4.6×250 mm)
and ope a ed using Empowe 2 so wa e. Mobile phase was
wa e :ace oni ile (95:5) a 1 mL/min and 35 ºC. The injec ion olume
was 50 µL and acyclo i was quan i ied a 251 nm ( e en ion ime 4.5
min). S anda d solu ions we e 0.075-15 µg/mL o acyclo i in wa e
(Volpa o e al., 1997). The cumula i e amoun s o acyclo i
pe mea ed pe a ea e sus ime we e i ed o a linea eg ession, and
he s eady s a e lux (J) and he lag ime ( lag) we e ob ained om he
slope and he x-in e cep o he linea eg ession, espec i ely (Al-
Ghabeish e al., 2015).
A e 6 h pe mea ion es , aliquo s o he dono chambe s we e
aken o subsequen analysis. Co neas/scle as we e imme sed du ing
24 h in 3 mL o e hanol:wa e (50:50 / ) medium, sonica ed du ing
99 min a 37 ºC, and cen i uged (1000 pm, 5 min, 25 ºC), and he
supe na an il e ed (Ac odisc® Sy inge Fil e , 0.22 µm GHP
Minispike, Wa e s), cen i uged again (14000 pm, 20 min, 25 ºC) and
il e ed o be measu ed in HPLC (Volpa o e al., 1997).
3.3. Resul s and Discussion
3.3.1. Micelles p epa a ion and cha ac e iza ion
Soluplus and Solu ol micelles we e p epa ed in wa e and PBS pH
7.4 using copolyme concen a ions up o 20% w/w. The pH o he
Soluplus solu ions in wa e was acid (pH 3.2) while Solu ol solu ions
ÁNGELA VARELA GARCÍA
92
we e nea ly neu al (pH 6.5). The di e en pH be ween copolyme
solu ions could a ec o he acyclo i solubili y (pKa 2.27 and 9.25).
CMCs o Soluplus and Solu ol we e epo ed o be 6.6x10-5 mM and
5.19x10-2 mM, espec i ely (BASF, 2010; BASF, 2012). The
concen a ions o each copolyme chosen o he s udy we e well
abo e he CMC alues, and anged be ween 0.09-1.74 mM o
Soluplus, and 10.38-207.63 mM o Solu ol (co esponding o 1-20%
copolyme concen a ion).
Size and Z-po en ial o micelles p epa ed wi h 12% copolyme a e
shown in Table 3.1. Soluplus micelles had an a e age size o 117.4
nm and a polydispe si y index o 0.23, while Solu ol micelles we e
much smalle showing an a e age size o 18.7 nm and a polydispe si y
index o 0.18. These alues a e in good ag eemen wi h li e a u e
(Hou e al., 2016). Loading o acyclo i caused a mino inc ease in
he size o Soluplus micelles (137.0 nm), bu ema kably inc eased he
size o Solu ol ones (134.9 nm). The su ace cha ge o bo h ypes o
copolyme was simila , being sligh ly nega i e o close o ze o.
3. Polyme ic micelles o acyclo i ocula deli e y: o mula ions and co nea
and scle a pe meabili y
93
Table 3.1. Size, polydispe sion index (PDI) and Z-po en ial o Soluplus and Solu ol
(12%) micelles in PBS pH 7.4 be o e and a e being loaded wi h acyclo i (ACV).
Mean alues and s anda d de ia ions in pa en hesis; n=3.
Fo mula ions Pa icle size (nm) PDI Z-po en ial (mV)
Soluplus 117.4 (1.4) 0.23 (0.01) -1.73 (0.94)
Solu ol 18.7 (4.0) 0.18 (0.02) -0.57 (0.38)
Soluplus + ACV 137.0 (4.0) 0.30 (0.02) 0.21 (0.85)
Solu ol + ACV 134.9 (1.4) 0.26 (0.02) -1.93 (0.54)
3.3.2. Rheological beha io
Solu ol (12 and 20%) dispe sions showed iscous-like beha io
wi h negligible G’ alues in he 15 o 40 ºC in e al (Figu e 3.2). The
iscosi y sligh ly dec eased wi h he inc ease in empe a u e. The
complex iscosi y alues eco ded we e in he ange 0.004 o 0.007
Pa.s, which is sligh ly highe han pu e wa e .
Di e en ly, Soluplus dispe sions showed a ema kable inc ease in
he alues o bo h G’ and G’’ when empe a u e su passes 30 ºC o 27
ºC in he case o 12 and 20% dispe sions, espec i ely (Figu e 3.2).
The inc ease was almos linea in he 30 o 37 ºC ange, and he G’’
alues a 37 ºC we e close o 7-40 Pa ( he highes alues we e
eco ded in PBS) and 1500 Pa o 12 and 20% dispe sions,
espec i ely. This beha io ag ees well wi h p e ious epo s on
Soluplus dispe sions (Al a ez-Ri e a e al., 2016) and i is qui e
di e en om ha showed by common in si u gelling copolyme

ÁNGELA VARELA GARCÍA
94
dispe sions ha exhibi a sha p inc ease in G’ and G’’ a he gelling
empe a u e. The complex iscosi y alues eco ded a 35 ºC o
Soluplus 12% in wa e and in PBS we e 0.50 and 3.12 Pa.s,
espec i ely, and o Soluplus 20% in wa e and in PBS we e 103.4,
and 79.7 Pa.s. These alues sugges ha he micelle o mula ions
could emain o p olonged ime on he ocula su ace.
3.3.3. Acyclo i solubiliza ion
Solubili y o acyclo i in wa e was 1.02 mg/mL, a alue sligh ly
lowe han he alues p e iously epo ed in li e a u e (Majumda e
al., 2009). Using sligh ly alkaline medium, such as PBS pH 7.4 o
SLF pH 7.5, he solubili y inc eased o 1.44 and 1.56 mg/mL,
espec i ely. This pa o he s udy was aimed o elucida e whe he
Soluplus and Solu ol micelles could encapsula e acyclo i , inc easing
i s appa en solubili y. In he ange o copolyme concen a ions
es ed, acyclo i solubili y posi i ely co ela ed wi h Soluplus
concen a ion. The enhancemen in solubili y was sligh ly highe in
PBS han in wa e , wi h appa en solubili y alues o 2.10 (s.d. 0.07)
and 2.05 (s.d. 0.04) mg/mL, espec i ely, in 20% w/w Soluplus
micelle medium.
3. Polyme ic micelles o acyclo i ocula deli e y: o mula ions and co nea
and scle a pe meabili y
95
Figu e 3.2. E ec o empe a u e on G’ and G’’ alues o Solu ol and Soluplus
dispe sions.
Solu ol
10 15 20 25 30 35 40 45
G' and G'' (Pa)
10
-3
10
-2
10
-1
Soluplus
Tempe a u e (ºC)
10 15 20 25 30 35 40 45
G' and G'' (Pa)
10
-3
10
-2
10
-1
10
0
10
1
10
2
10
3
10
4
10
5
G' G'', 12% in wa e
G' G'', 12% in PBS
G' G'', 20% in wa e
G' G'', 20% in PBS
G'', 12% in wa e
G'', 12% in PBS
G'', 20% in wa e
G'', 20% in PBS
ÁNGELA VARELA GARCÍA
96
Di e en ly, acyclo i solubili y in 20% w/w Solu ol micelles showed
a mino inc ease, being 1.54 (s.d. 0.03) and 1.39 (s.d. 0.06) mg/mL in
PBS and wa e , espec i ely (Figu e 3.3).
Figu e 3.3. Appa en solubili y o acyclo i in micelle dispe sions o Soluplus and
Solu ol HS 15, p epa ed in wa e and PBS pH 7.4. E o ba s ep esen s s anda d
de ia ion (n=3).
Ne e heless, compa ed o o he d ugs o mula ed in hese
polyme ic micelles, he o al inc ease in solubili y was ela i ely low,
wi h may be ela ed o he pola i y o acyclo i (LogP= -1.56) (Al-
Ghabeish e al., 2015; Hou e al., 2016).
F ee ene gy o solubiliza ion was nega i e in all cases, which
means ha he solubiliza ion occu ed spon aneously and was
he modynamically a o ed by he hyd ophobici y o micelle co e.
P e ious wo ks ha e epo ed on solubiliza ion alues o up o 15
Copolyme concen a ion (% w/w)
0 4 8 12 16 20
Acyclo i solubilized (mg/mL)
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
Soluplus-Wa e
Soluplus-PBS
Solu ol-Wa e
Solu ol-PBS
3. Polyme ic micelles o acyclo i ocula deli e y: o mula ions and co nea
and scle a pe meabili y
97
mg/ml using O’O-lau oyl chi osan (Tan, 2016). Despi e o ha ing
simila HLB alues, Soluplus composi ion and a chi ec u e as a sel -
assembled copolyme seem o be mo e sui able o hos acyclo i han
he Solu ol mix u e o poly (e hylene oxide) es e s o 12-
hyd oxys ea ic acid and ee polye hylene glycol. The e o e, Solu ol
was disca ded o subsequen s udies.
Pa ame e s used o quan i y he e iciency o solubiliza ion a e
summa ized in Table 3.2. The micelle-wa e pa i ion coe icien (P)
o Solu ol dispe sions was below 1, which means ha he e a e mo e
acyclo i molecules solubilized in he aqueous medium han inside he
micelles. Di e en ly, an inc ease in he pa i ion coe icien alues
was obse ed o Soluplus as he copolyme concen a ion inc eased,
indica ing ha o micelle sys ems p epa ed wi h Soluplus a 16% o
highe he amoun o acyclo i in o he micelles su passes he amoun
solubilized in he ou e aqueous medium.
ÁNGELA VARELA GARCÍA
104
Figu e 3.6. Accumula ed amoun s o acyclo i pe mea ed h ough bo ine scle a
du ing 6 h, o aqueous solu ion o ACV (0.3 mg/mL) and o mula ion o ACV in
Soluplus 20% micelles (1.34 mg/mL), a 37 ºC.
The esul s showed 10 imes g ea e amoun s o d ug pe mea ed
h ough he scle a han h ough he co nea. Since he expe imen s
we e ca ied using simila su ace a ea o bo h issues, he di e ences
a e clea ly ela ed o he g ea e pe meabili y o scle a, which has a
ela i ely po ous s uc u e ha allows d ug di usion ei he as ee
molecules o a e being encapsula ed in micelles (Ahmed e al., 1987;
Hamalainen e al., 1997; Tai e al., 2003; Wen e al., 2010; Wen e al.,
2013). P e ious s udies wi h cyclospo ine encapsula ed in
Plu onic/TPGS mixed micelles showed ha he d ug accumula ed in
he scle a (p obably because hyd ophobic in e ac ions) and only a
small ac ion pe mea ed h ough he ecep o (G imaudo e al., 2018).
Di e en ly, in he case o acyclo i -Soluplus micelles, he e was also
Time (h)
0123456
Acyclo i pe mea ed (g/cm
2
)
0
20
40
60
80
100
120
140
160
180
ACV
ACV-Soluplus

3. Polyme ic micelles o acyclo i ocula deli e y: o mula ions and co nea
and scle a pe meabili y
105
scle a accumula ion bu mos d ug could eadily di use h ough i ,
which may a o he access o he pos e io eye segmen . Compa ed o
acyclo i ee in solu ion, encapsula ion in Soluplus micelles was
ad an ageous bo h in e ms o (i) o al amoun o acyclo i
accumula ed in he scle a, which was 2.02 µg/cm2 (s.d. 1.61) when
applied as aqueous solu ion, and 13.69 µg/cm2 (s.d. 4.10) when
o mula ed in Soluplus micelles; and (ii) s eady s a e lux (J) ob ained
om he slope o he amoun pe mea ed h ough he scle a s. ime,
which was 8.02 and 26.97 µg/cm2·h-1 o acyclo i aqueous solu ion
and micelle o mula ion, espec i ely.
3.4. Concluding ema ks
Soluplus micelles loaded wi h acyclo i showed a homogeneous
nanome e pa icle size wi h sligh ly nega i e Z-po en ial alues,
which may be adequa e o c oss co nea and scle a. In addi ion, his
sys em was shown o be sui able o o mula ion as eye d ops ha may
unde go in si u gelling since he s o age and loss moduli inc ease as
empe a u e aise om oom empe a u e o ocula empe a u e. The
inc ease in iscosi y o he o mula ion would p olong i s pe manence
on he eye su ace, slowing down he dilu ion p ocess. The
encapsula ion o he an i i al d ug did no g ea ly imp o e i s appa en
solubili y, bu i did no ably acili a e he pene a ion h ough and
accumula ion in he eye issues. In pa icula , he no iceable
accumula ion o acyclo i a he scle a le el may acili a e he access
o his d ug o he pos e io segmen o he eye.
ÁNGELA VARELA GARCÍA
106
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342
Imp in ed hyd ogels o ocula adminis a ion o
acyclo i and alacyclo i
Imp in ed
hyd ogels
0.0
2.0
4.0
6.0
0 5 10 15 20 25
VACV eleased
(mg/g)
Time (h)
MIP
-
1:12
ÁNGELA VARELA GARCÍA
120
The aim o he second s ep o he Thesis was o design hyd ogels
sui able o so CL wi h a ini y o acyclo i and alacyclo i and
ha can sus ainedly elease hese d ugs on he ocula su ace du ing
daily use. Among he p oposed p ocedu es o endow he hyd ogel CLs
wi h 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 (By ne e al.,
2002; Al a ez-Lo enzo e al., 2004). This echnique equi es
inco po a ing he subs ances o in e es 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 subs ance o in e es
again (Al a ez-Lo enzo e al., 2004). Func 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; me hac ylic acid (MAA) showed 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 (Figu e 4.1).
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. 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 he d ug. The
hyd ogels will be cha ac e ized in e ms o swelling, ansmi ance,
mechanical p ope ies, biocompa ibili y (HET-CAM assay) and

4. Imp in ed hyd ogels o acyclo i and alacyclo i ocula adminis a ion
121
capabili y o load and elease he an i i al d ugs. Finally, he
pe meabili y h ough bo ine co nea and scle a o he d ug eleased by
he hyd ogel was e alua ed.
Figu e 4.1. S uc u e o me hac ylic acid, acyclo i and alacyclo i .
4.2. Ma e ials and me hods
4.2.1. Ma e ials
Acyclo i was pu chased om Fa malabo (I aly); alacyclo i ,
2,2′-azo-bis(isobu y oni ile) (AIBN), dichlo odime hylsilane,
e hyleneglycol dime hac yla e (EGDMA) and me hac ylic acid
(MAA) we e om Sigma-Ald ich (Ge many); e hanol absolu e and
NaOH we e om VWR (Belgium); 2-hyd oxye hyl me hac yla e
(HEMA) was om Me ck (Ge many); ace ic acid and NaCl we e om
Scha lau (Spain); and me hanol was om Fishe (Belgium). Ul apu e
wa e ( esis i i y > 18 MΩ·cm) was ob ained by e e se osmosis
(MilliQ®, Millipo e 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
NaHCO3, 1.38 g/L KCl and 0.084 g/L CaCl2·2H2O wi h pH 7.5.
Ca bona e bu e pH 7.2 was p epa ed by mixing bu e solu ion A
Me hac ylic acid
Valacyclo i
Acyclo i
ÁNGELA VARELA GARCÍA
122
(6.2 g/L NaCl, 0.355 g/L KCl, 0.1 g/L NaH2PO4·H2O and 2.45 g/L
NaHCO3) and bu e solu ion B (0.115 g/L CaCl2 and 0.155 g/L
MgCl2·6H2O).
4.2.2. 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 yl amide (AAm), 2-aminoe hyl
me hac yla e hyd ochlo ide (AEMA), N-(3-aminop opyl)
me hac ylamide hyd ochlo ide (APMA), e hyleneglycolphenyle 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 (MAAc).
The 3D s uc u e o he unc ional monome s and ACV and VACV
was aken om he PubChem da abase (Kim e al., 2016). The
Au odock Tools e sion 4.2.6 so wa e was used o calcula e
molecula docking. In all 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 (Mo is e al., 2009). Es ima ed ee ene gy o
binding (

Gbinding) and dissocia ion cons an (Ki) alues we e
ob ained.
4.2.3. Syn hesis o imp in ed hyd ogels
Di e en mix u es o monome s we e p epa ed, as shown in
Table 4.1. The componen s we e added o ials and mixed a oom
4. Imp in ed hyd ogels o acyclo i and alacyclo i ocula adminis a ion
123
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 minu es mo e. The solu ions we e
injec ed, wi h needle and sy inge, in o p e-assembled moulds,
consis ing o wo p e- ea ed glass pla es (12x14 cm) sepa a ed by a
0.45 mm hick silicone ame. The p e- ea men o he glass consis ed
o applying wo laye s o dichlo odime hylsilane, wai ing 10 minu es
be ween each applica ion. The pla es we e le o d y in a hood o 1
hou and hen we e ho oughly washed wi h e hanol and insed wi h
wa e . Finally, hey we e d ied in an o en a 70ºC o 1 hou , be o e
being assembled. Polyme iza ion was ca ied ou o 12 hou s a 50°C
and hen o a u he 24 hou s a 70°C. All hyd ogel composi ions
we e p epa ed in iplica e.
ÁNGELA VARELA GARCÍA
124
Table 4.1. Composi ion o he syn hesized hyd ogels (NIP: non-imp in ed hyd ogels,
MIP: imp in ed hyd ogels). Final EGDMA, MAA and 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)
NIP0 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
MIP1:5 5 7.55 0.084 45 0 8.21
MIP1:10 5 7.55 0.084 23 0 8.21
MIP1:15 5 7.55 0.084 15 0 8.21
MIPVACV 5 7.55 0 0 25 8.21
MIP1:6 5 7.55 0.084 0 50 8.21
MIP1:12 5 7.55 0.084 0 25 8.21
MIP1:32 5 7.55 0.084 0 10 8.21
4.2.4. D ug emo al
A e polyme iza ion, each hyd ogel shee was imme sed in 500
mL o boiling wa e o 15 minu es in o de o emo e un eac ed
monome s and acili a e cu ing in o discs (10 mm in diame e ).
Washing was hen pe o med, excep o a ew discs o each ype o
hyd ogel, which we e ese ed o di ec d ug elease es , as
explained below. 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
media was eplaced e e y 24 hou s un il no signal was measu ed,
which was moni o ed spec opho ome ically in he ange o 190-800
nm (UV-Vis spec opho ome e , Agilen 8534, Ge many). When no
4. Imp in ed hyd ogels o acyclo i and alacyclo i ocula adminis a ion
125
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 hou s and s o ed p o ec ed om ligh and
humidi y. In pa allel, he amoun o ACV (MIPACV, MIP1:5, MIP1:10
and MIP1:15) and VACV (MIPVACV, MIP1:6. MIP1:12 and MIP1:32)
emo ed in each washing s ep was moni o ed spec opho ome ically
a 252 and 253 nm, espec i ely.
4.2.5. Di ec d ug elease es om boiled hyd ogels
Discs o each ype o hyd ogel we e indi idually placed in 5 mL
o SLF and kep unde oscilla ing agi a ion (300 pm) and a 35 ºC. A
p ese imes (0.5, 1, 2, 6 and 24 h), 3 mL o medium we e emo ed
and abso bance was measu ed by spec opho ome y a 252 nm
(ACV) and 253 nm (VACV) (UV-Vis spec opho ome e , Agilen
8453, 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 he 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 µg/mL) in e hanol:wa e
(50:50, / ) mix u e, and VACV (50 µg/mL) in wa e . Dilu ions o 2,
3, 5, 10, 15, 20, 25 and 30 µg/mL we e made o ACV, and 1.25, 2.5,
5, 10, 15, 20, 25, 30, 40 and 50 µg/mL o VACV. The calib a ion
cu e was p epa ed om abso bances eco ded a 252 and 253 nm,
espec i ely (UV-Vis spec opho ome e , Agilen 8453, Ge many).

ÁNGELA VARELA GARCÍA
126
4.2.6. D ug loading and elease om condi ioned hyd ogels
The ACV load was es ed on he hyd ogels NIP0, NIP200, MIPACV,
MIP1:5, MIP1:10 and MIP1:15. Washed and d y discs o each ype we e
placed in iplica e in ubes wi h 15 mL o d ug solu ion. The loading
solu ion was p epa ed by dissol ing ACV (0.3 mg/mL) in wa e and
kep unde magne ic agi a ion (300 pm), a oom empe a u e o 72h.
The loading ubes we e kep unde oscilla ing agi a ion (300 pm), a
RT (23-25 ºC), o 4 days. The abso bances o each sample we e
measu ed by spec opho ome y a 252 nm (UV-Vis
spec opho ome e , Agilen 8453, Ge many), dilu ing he sample
(0.2:5) wi h e hanol:wa e mix u e (50:50, / ). The calcula ion o he
o al d ug load on he discs was es ima ed by he di e ence be ween
he ini ial and inal amoun o d ug in solu ion, and was calcula ed
using he p e iously p epa ed calib a ion cu e, e e ing he loaded
amoun s o he uni o mass o he d y disc.
The VACV load was e alua ed on he hyd ogels NIP0, NIP200,
MIPVACV, MIP1:6, MIP1:12 and MIP1:32. Th ee d y discs o each ype
we e placed in ubes wi h 5 mL o d ug solu ion. The loading solu ion
was p epa ed by dissol ing VACV (0.3 mg/mL) in a 0.1 mM NaOH
solu ion and was kep unde magne ic agi a ion (300 pm), a RT (23-
25 ºC), o 15 minu es. 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. The
abso bances o each sample we e measu ed spec opho ome ically, a
253 nm, o he calcula ion o he o al load, in he same way.
4. Imp in ed hyd ogels o acyclo i and alacyclo i ocula adminis a ion
127
The d ug ne wo k/wa e pa i ion coe icien (KN/W) was
calcula ed o each hyd ogel om he o al amoun o d ug loaded
using he ollowing equa ion:
𝐿𝑜𝑎𝑑𝑖𝑛𝑔 (𝑡𝑜𝑡𝑎𝑙)=
∗
∗𝐶 [Eq. 4.1]
whe e Vs is he olume o wa e abso bed by he hyd ogel, Vp he
olume o d y polyme , Wp he weigh o he d y hyd ogel, and C0 he
concen a ion o d ug in he loading solu ion.
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 hen he
discs we e 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 ºC. The elease kine ics we e e alua ed a 1, 2, 4,
6, 8 and 24 h, o ACV, and a 0.5, 1, 2, 3, 4, 5, 6, 10 and 24 h, o
VACV. The samples we e aken ollowing he same p o ocol as in
sec ion 4.1.4.
4.2.7. Deg ee o swelling
The deg ee o swelling was moni o ed in wa e and SLF o
hyd ogels NIP0, NIP200, MIPVACV, MIP1:6, MIP1:12 and MIP1:32, in
iplica e. The s udy was ca ied ou a RT (23-25 ºC) and he
inc emen in weigh o he hyd ogels was eco ded a p ede e mined
imes (0.5, 1, 2, 4, 8 and 24 h), a e being subme ged in 4 mL o he
co esponding medium. In each measu emen , he disc was emo ed
om he ial, supe icially d ied wi h il e pape , weighed and
ÁNGELA VARELA GARCÍA
128
e u ned o he ial. The deg ee o swelling was calcula ed wi h he
ollowing equa ion:
𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑑𝑒𝑔𝑟𝑒𝑒 (%)=
∗100 [Eq. 4.2]
whe e W0 and W ep esen he weigh o he d ied and swollen
hyd ogel, espec i ely.
4.2.8. Ligh ansmi ance
The ligh ansmission (%) o he hyd a ed discs in he swelling
es (SLF) was measu ed in a spec opho ome e (Agilen Ca y 60
UV-Vis) in iplica e, eco ding he ansmi ance om 200 o 800 nm.
4.2.9. Mechanical p ope ies
The mechanical p ope ies o he NIP0, NIP200, MIPVACV, MIP1:6,
MIP1:12 and MIP1:32 discs, swollen in wa e , we e es ed in iplica e a
RT (23-25 ºC). Each hyd ogel was cu in o 16 x 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., UK), equipped
wi h a 5 kg load cell. The c osshead speed which he s ess-s ain
plo s we e eco ded was 0.1 mm s-1. Fo he calcula ion o Young's
modulus (E), he slope o he s aigh line pa o he ensile s eng h
( o ce pe c oss-sec ional a ea) and he enginee ing s ess (change o
ac i e leng h di ided by o iginal leng h) we e used, as ollows
(T anoudis e al., 2004; Bham a e al., 2017).
𝐸 = 

∆ 
 [Eq. 4.3]
4. Imp in ed hyd ogels o acyclo i and alacyclo i ocula adminis a ion
129
4.2.10. HET-CAM es
The cho ioallan oic memb ane hen egg es (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) (Al a ez-Ri e a e al., 2019).
The es was pe o med by placing in each CAM a hyd ogel o
each ype in iplica e, p e iously hyd a ed o 24 h in loading
solu ion. An aqueous solu ion o NaOH 0.1N and NaCl 0.9% (300
µL) in iplica e was 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.
4.2.11. Bo ine co neal and scle al pe meabili y es
The esh bo ine eyes we e collec ed om he local
slaugh e house and anspo ed acco ding o he BCOP es p o ocol
(OECD, 2009; Al a ez-Ri e a e al., 2019). 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 μg/ml), in an ice ba h. The 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
ÁNGELA VARELA GARCÍA
232

7. Pa en
233
ÁNGELA VARELA GARCÍA
234
7. Pa en
235
ÁNGELA VARELA GARCÍA
236
Abb e ia ions

8. Abb e ia ions
239
8. Abb e ia ions
 AAPH 2,2′-azobis(2-amidino-p opane) dihyd ochlo ide
 ACV Acyclo i
 AFR A ica
 AIBN 2,2′-azo-bis(isobu y oni ile)
 AMD Age- ela ed macula degene a ion
 AMR Ame ica
 BAB Hema o-aqueous ba ie
 BCOP Bo ine co nea opaci y es
 BRB Hema o- e inal ba ie
 CAT Ca alase
 CD Cyclodex ins
ÁNGELA VARELA GARCÍA
240
 CLs Con ac lenses
 CMC C i ical micella concen a ion
 CMV Ci omegalo i us
 CO Co neal opaci ies
 DES D y eye synd ome
 DME Macula edema
 DR Diabe ic e inopa hy
 EDTA E hylenediamine e aace ic acid
 EGPEM E hylene glycol-phenyl e he me hac yla e
 EMR Eas e n Medi e anean
 EUR Eu ope
 GMA Glycidyl me hac yla e
 GPx Glu a hione pe oxidase
 HCEC Human co neal epi helial cells
 HEMA Hyd oxye hyl me hac yla e
8. Abb e ia ions
241
 HET-CAM Hen’s egg es on cho io-allan oic memb ane
 HLB Hyd ophilic-lipophilic balance
 HSV He pes simplex i us
 IOP In aocula p essu e
 MAA Me hac ylic acid
 MW Molecula weigh
 ORAC Oxygen adical an ioxidan capaci y
 PBS Phospha e bu e ed saline
 PDI Polydispe sion index
 PLGA Poly lac ic-co-glycolic acid
 PVR P oli e a i e i eo e inopa hy
 RE Re ac i e e o s
 RNS Reac i e ni ogen species
 ROS Reac i e oxygen species
 RT Room empe a u e