Strategies for ocular administration of antiviral and antioxidant agents

DOCTORAL THESIS STRATEGIES FOR OCULAR ADMINISTRATION OF ANTIVIRAL AND ANTIOXIDANT AGENTS Ángela Varela García 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 Varela García ESCUELA DE DOCTORADO INTERNACIONAL PROGRAMA DE DOCTORADO EN INVESTIGACIÓN Y DESARROLLO DE MEDICAMENTOS SANTIAGO DE COMPOSTELA 2020 AUTHORIZATION OF THE THESIS SUPERVISORS Strategies for ocular administration of antiviral and antioxidant agents Prof. Carmen Alvarez Lorenzo Prof. Angel Concheiro Nine REPORT: That the present Thesis, corresponds to the work carried out by Miss Ángela Varela García, under our supervision, and that we authorize its presentation considering it gathers the necessary requirements of article 34 of the USC Doctoral Studies Regulation, and that as supervisors of this Thesis, it does not incur in the abstention causes established by the law 40/2015 At Santiago de Compostela, on June 8 th 2020 Prof. Carmen Alvarez Lorenzo Prof. Angel Concheiro Nine AUTORIZACIÓN DEL DIRECTOR / TUTOR DE LA TESIS Estrategias para la administración ocular de fármacos antivirales y antioxidantes Prof. Carmen Alvarez Lorenzo Prof. Angel Concheiro Nine INFORMAN: Que la presente Tesis, se corresponde con el trabajo realizado por Dña. Ángela Varela García, bajo nuestra supervisión y autorizamos su presentación , considerando que reúne l o s r equisitos exigidos en la Regulación de Estudios de Doctorado de la USC, y que como directores de ésta no incurre en las causas de abstención establecidas e n la Ley 40/2015. En Santiago de Compostela, a 8 de Junio de 2020 Prof. Carmen Alvarez Lorenzo Prof. Angel Concheiro Nine PhD CANDIDATE STATEMENT Strategies for ocular administration of antiviral and antioxidant agents Miss Ángela Varela García I submit my Doctoral Thesis, following the procedure according to the Regulation, stating that: 1) This Thesis gathers the results corresponding to my work. 2) When necessary, explicit mention is given to the collaborations the work may have had. 3) The present document is the final version submitted for its defense and coincides with the document sent in electronic format. 4) I confirm that the Thesis does not incur in any plagiarism of any other authors or documents submitted by me for obtaining other degrees. At Santiago de Compostela, on June 8 th 2020 Sgd. Ángela Varela García AGRADECIMIENTOS Cuando una etapa tan importante de tu vida llega a su fin, es inevitable echar la vista atrás para hacer balance de todo lo positivo que ha ocurrido e influido en tu crecimiento personal y profesional. Son muchas las personas que han aportado algo durante este largo trayecto, las cuales formarán parte de esta historia para siempre. Por ello, me gustaría agradecer a todos los que de una forma u otra han estado presentes en ella. A mis directores de tesis, los profesores Carmen Alvarez Lorenzo y Angel Concheiro Nine. Estaré eternamente agradecida por haberme dejado formar parte de vuestro equipo. Me habéis enseñado que, con esfuerzo y perseverancia, todo se puede conseguir. Gracias por todo lo aprendido. A los profesores del Departamento de Farmacología, Farmacia y Tecnología Farmacéutica, José Luis Gómez Amoza, Carlos García González, Mariana Landín Pérez, Loli Torres López, Carmen Remuñán López, y, en especial, a Francisco Otero Espinar, siempre dispuestos a prestar ayuda en todo lo necesario. A los profesores Aisling Ní Annaidh, de School of Mechanical and Materials Engineering de la UCD de Dublín, y Lorenzo Pastrana, del International Iberian Nanotechnology Laboratory de Braga, por haberme dado la oportunidad de seguir formándome y de ampliar mis conocimientos desde otro punto de vista. A los ya doctores, Ale, Sonia, Patri, Isa y Luis, por darme la bienvenida a esta familia y por ayudar a resolver siempre las dudas de una novata. En especial a Fer, que pasó de ser mentor a amigo, por haber pasado tantas horas explicándome y ayudándome, por todas las risas y cabreos, y por el apoyo constante. A mis colegas de máster, Laura, Lucía, Ana y Diego, por haber compartido tantos momentos, por esas charlas infinitas que nunca son suficiente y por todo el apoyo mutuo. Al “Team Fran”, Victoria, Andrea Conde, Xurxo, Rubén, Guille, Iria, Carlos y Andrea Luaces, por esa locura contagiosa que, sin duda, hace que el día a día sea mucho más que agradable. A mis compañeros de batalla. A María, porque estoy convencida de que sin ti nada sería igual, porque tu generosidad no tiene límites y por haber forjado esta bonita amistad que será para siempre. A Xián, por todas las conversaciones, risas y llantos compartidos. A los vecinos de arriba, Clara, Viti, Rebeca y Ana, por ser tan buenos compañeros y por haber pasado juntos tantos buenos momentos, así como a Lorena y a Helena. A las incorporaciones más recientes, Ana Filipa, Iago, Axel y Patri porque aunque llevemos poco tiempo juntos, el compañerismo y el buen ambiente que tenemos es en parte gracias a vosotros. A los que ya se han ido, Mirian y Mariano, por haber estado siempre dispuestos a ayudar sin dudarlo. Y a los que estuvieron de paso, pero dejaron huella, a Anna Paula, Catia y Adrián, porque en poco tiempo habéis pasado a formar parte de esta gran familia y, en especial, a Claudia, por todos los buenos momentos compartidos y por esta amistad que nos une. A Andrea, por haber sido una compañera de trabajo excepcional, por todos los buenos momentos pasados y por todos los que vendrán. A Sole. Resulta complicado expresar en pocas palabras lo agradecida que estoy por todo lo que haces. Jefa, compañera, mentora y amiga, siempre dispuesta a ayudar en todo. Ha sido un placer y un privilegio haber podido compartir esta etapa de mi vida contigo. A todos mis amigos y a mi familia, en especial a Rocío y Vanesa, mis “hermanas” mayores; también a Diana, Cores y Sofía, por estar siempre ahí. A Marcos, mi compañero de vida, cuya paciencia infinita y apoyo incondicional hacen que pueda mantenerme siempre a flote. Por último, a las personas más importantes de mi vida, Pilar y Ricardo, mis padres, ejemplos máximos de superación y valentía, que me han sabido enseñar lo más valioso de la vida, nunca rendirse y no temer a las dificultades. No hay palabras suficientes para agradecer todo el apoyo recibido en cada momento, sin el cual nunca llegaría a ser la persona que hoy en día soy. A todos, de corazón, mil gracias. Index Resumen ........................................................................................................ 3 1. Introduction ........................................................................................... 19 1.1. Ocular anatomy ................................................................................ 22 1.2. Eye-defense mechanisms ................................................................. 26 1.2.1. Lacrimal film .............................................................................. 27 1.2.2. Corneal barrier ........................................................................... 28 1.2.3. Non-corneal barriers ................................................................... 29 1.2.4. Blood ocular barriers .................................................................. 30 1.3. Ocular drug administration ............................................................ 31 1.4. Cornea diseases ................................................................................ 34 1.4.1. Ocular trauma ............................................................................. 35 1.4.2. Degenerative disorders ............................................................... 36 1.4.3. Inflammatory diseases ................................................................ 38 1.4.4. Infectious diseases ...................................................................... 39 1.4.5. Prevention and treatment of corneal lesions: antioxidant agents 39 1.5. Ocular viral infections ..................................................................... 41 1.5.1. Treatment of ocular viral infections ........................................... 44 1.6. Micelles and contact lenses as platforms for ocular delivery ....... 46 1.6.1. Polymeric micelles ..................................................................... 46 1.6.2. Contact lenses ............................................................................. 50 1.7. References ......................................................................................... 58 2. Aims ........................................................................................................ 73 3. Polymeric micelles for acyclovir ocular delivery: formulation and cornea and sclera permeability ................................................................. 81 3.1. Introduction ...................................................................................... 81 3.2. Materials and methods ..................................................................... 86 3.2.1. Materials ..................................................................................... 86 3.2.2. Micelles preparation and characterization .................................. 87 3.2.3. Rheological behavior .................................................................. 87 3.2.4. Acyclovir solubilization ............................................................. 88 3.2.5. Micelle stability against dilution ................................................ 89 3.2.6. Corneal and sclera permeability assay ........................................ 90 3.3. Results and Discussion ..................................................................... 91 3.3.1. Micelles preparation and characterization .................................. 91 3.3.2. Rheological behavior .................................................................. 93 3.3.3. Acyclovir solubilization ............................................................. 94 3.3.4. Micelle stability against dilution ................................................ 99 3.3.5. Cornea and sclera permeability assay ....................................... 100 3.4. Concluding remarks ....................................................................... 105 3.5. References ....................................................................................... 106 4. Imprinted hydrogels for acyclovir and valacyclovir ocular administration ........................................................................................... 115 4.1. Introduction .................................................................................... 115 4.2. Materials and methods ................................................................... 121 4.2.1. Materials ................................................................................... 121 4.2.2. Computational modeling .......................................................... 122 4.2.3. Synthesis of imprinted hydrogels ............................................. 122 4.2.4. Drug removal ............................................................................ 124 4.2.5. Direct drug release test from boiled hydrogels ......................... 125 4.2.6. Drug loading and release from conditioned hydrogels ............. 126 4.2.7. Degree of swelling .................................................................... 127 4.2.8. Light transmittance ................................................................... 128 4.2.9. Mechanical properties .............................................................. 128 4.2.10. HET-CAM test ................................................................... 129 4.2.11. Bovine corneal and scleral permeability test ...................... 129 4.3. Results and Discussion ................................................................... 131 4.3.1. Computational modeling .......................................................... 131 4.3.2. Synthesis of hydrogels and drug removal ................................ 133 4.3.3. Direct drug release test from boiled hydrogels ........................ 134 4.3.4. Drug loading in washed hydrogels ........................................... 136 4.3.5. Drug release ............................................................................. 138 4.3.6. Hydrogel characterization ........................................................ 140 4.3.7. HET-CAM test ......................................................................... 142 4.3.8. Bovine corneal and scleral permeability test ............................ 143 4.4. Concluding remarks ...................................................................... 146 4.5. References ....................................................................................... 147 5. Cytosine-functionalized bioinspired hydrogels for ocular delivery of antioxidant transferulic acid ................................................................... 155 5.1. Introduction .................................................................................... 155 5.2. Materials and methods .................................................................. 161 5.2.1. Materials ................................................................................... 161 5.2.2. Hydrogel synthesis ................................................................... 162 5.2.3. Functionalization with cytosine ............................................... 163 5.2.4. Hydrogels characterization ....................................................... 164 5.2.5. TA loading ............................................................................... 166 5.2.6. TA release ................................................................................ 166 5.2.7. HET-CAM test ......................................................................... 167 5.2.8. Cytocompatibility assay ........................................................... 167 5.2.9. Antioxidant activity .................................................................. 168 5.2.10. Cornea and sclera penetration and accumulation ............... 169 5.3. Results and discussion ................................................................... 171 5.3.1. Hydrogels synthesis and cytosine grafting ............................... 171 5.3.2. Swelling, light transmission and mechanical properties ........... 175 5.3.3. TA loading ................................................................................ 177 5.3.4. TA release ................................................................................. 180 5.3.5. Biocompatibility ....................................................................... 182 5.3.6. Antioxidant activity .................................................................. 183 5.3.7. Cornea and sclera penetration................................................... 185 5.4. Concluding remarks ....................................................................... 189 5.5. References ....................................................................................... 190 6. Conclusions .......................................................................................... 201 7. Patent .................................................................................................... 207 8. Abbreviations ....................................................................................... 239 Resumen ÁNGELA VARELA GARCÍA 8 visión borrosa, enrojecimiento o lagrimeo. La queratitis por herpes simple (infección e inflamación de la córnea) es la principal causa de ceguera por infección a nivel mundial. El aciclovir penetra en las células infectadas y compite con los nucleósidos naturales para incorporarse al ADN y poder actuar como terminador de cadena. Después de anclarse, da lugar a un mecanismo de inactivación suicida, puesto que el ADN terminado se une a la polimerasa del ADN viral y la inhibe de manera irreversible, impidiendo la replicación del virus. El tratamiento tópico clásico requiere un periodo de aplicación prolongado y da lugar a efectos secundarios graves. La administración oral se caracteriza por presentar una biodisponibilidad inferior al 20%, por lo que son necesarias dosis muy altas durante tiempos prolongados. La hipótesis de esta primera etapa de la Tesis fue que la encapsulación de aciclovir en nanomicelas poliméricas debe aumentar la solubilidad del fármaco y promover su acumulación en córnea y esclera y el acceso a estructuras más profundas. Para llevar a cabo el trabajo, primero se hizo un barrido de las prestaciones de los copolímeros Soluplus y Solutol. Se prepararon dispersiones de cada copolímero cubriendo un amplio intervalo de concentraciones (1, 4, 8, 12, 16 y 20% p/p) en agua y tampón fosfato (PBS) pH 7.4, y se evaluó su tamaño, potencial Z y comportamiento reológico. Las propiedades viscoelásticas se caracterizaron a partir de medidas de los módulos de almacenamiento (G’) y de pérdida (G’’) de Resumen 9 las dispersiones de Soluplus y Solutol al 12% y al 20%, en función de la temperatura (15-40 °C). Las dispersiones de Solutol mostraron un comportamiento viscoso. En cambio, las dispersiones de Soluplus mostraron incrementos muy marcados en G’ y G’’ a temperaturas próximas a la corporal. La capacidad de las nanomicelas para solubilizar aciclovir se evaluó en agua, PBS pH 7.4 y fluido lacrimal artificial (FLS) pH 7.5. Se calcularon parámetros de solubilidad como la capacidad de solubilización molar, el coeficiente de reparto micelaagua y la energía de solubilización libre de Gibbs. Únicamente las micelas de Soluplus dieron lugar a incrementos relevantes de la solubilidad de aciclovir, mostrando una elevada capacidad de encapsulación y estabilidad frente a la dilución. Finalmente, se llevaron a cabo ensayos de permeabilidad a través de córnea y esclera bovinas en células de difusión. En comparación con el aciclovir libre en disolución, la encapsulación en micelas de Soluplus dio lugar a marcados aumentos en la cantidad de fármaco acumulada en ambos tejidos, así como en la cantidad que pasa al compartimento receptor. En suma, las micelas de Soluplus presentaron propiedades fisicoquímicas adecuadas para la administración ocular de aciclovir, mejorando la penetración del fármaco a través de córnea y esclera. 2) Diseño de hidrogeles imprinted para fármacos antivirales. Con el fin de prolongar la permanencia del fármaco en la superficie ocular más allá de lo que permiten las formulaciones en nanomicelas, el ÁNGELA VARELA GARCÍA 10 objetivo de la segunda etapa de la Tesis fue diseñar hidrogeles válidos para LC blandas con afinidad por aciclovir y valaciclovir. Entre los procedimientos propuestos para dotar a los hidrogeles de afinidad por moléculas específicas, destaca la creación de receptores artificiales utilizando la técnica de moldeado molecular (molecular imprinting). Esta técnica requiere incorporar la sustancia de interés a la mezcla de monómeros para que estos se reordenen en función de su afinidad, y el reordenamiento se haga permanente durante la polimerización. La remoción de las moléculas molde genera cavidades con el tamaño y los grupos químicos más adecuados para alojar de nuevo la sustancia de interés. La selección del ácido metacrílico (MAA) como monómero funcional se hizo teniendo en cuenta que, además de ser un monómero habitual en la preparación de LC, en un estudio preliminar por modelización computacional mostró capacidad para interaccionar tanto con el anillo de aciclovir y valaciclovir como con el grupo amino de la cadena lateral valaciclovir. Se sintetizaron hidrogeles mezclando HEMA con MAA como monómero funcional, y se añadió aciclovir o valaciclovir en distintas proporciones. Tras la polimerización, se confirmó la extracción del fármaco durante los lavados mediante espectrofotometría UV. Con algunos hidrogeles se llevó a cabo un estudio de cesión directa del fármaco (sin lavado previo), para determinar la capacidad de control de la cesión del fármaco incorporado durante la polimerización. Una Resumen 11 vez lavados los hidrogeles, se estudió su capacidad de reincorporación de aciclovir o valaciclovir, por inmersión en disoluciones acuosas de cada fármaco. Se comprobó que los discos presentaban mayor afinidad por valaciclovir. A continuación, se llevó a cabo un estudio de cesión con ambos fármacos en FLS, lo que confirmó que los hidrogeles impresos y cargados con valaciclovir presentaban perfiles de cesión más adecuados, con una liberación sostenida durante 10 h que da lugar a niveles terapéuticos. Los hidrogeles se caracterizaron en términos de hinchamiento, transmitancia y propiedades mecánicas, obteniéndose valores similares a los que son comunes para las LC hidrófilas. También se sometieron a un ensayo de irritación en membrana corioalantoidea de huevo (HET-CAM), no observándose hemorragia, lisis ni coagulación. Finalmente, con los hidrogeles con mejores propiedades de carga y cesión de valaciclovir se llevaron a cabo ensayos de permeabilidad en córnea y esclera bovinas. De la misma forma que en el caso de la disolución acuosa de valaciclovir, el fármaco cedido por los hidrogeles fue capaz de acumularse en la cornea y de penetrar a través de la esclera. Los resultados obtenidos indican que los hidrogeles preparados con MAA como monómero funcional e impresos con valaciclovir son buenos candidatos para la administración tópica ocular de este fármaco. 3) Diseño de hidrogeles bioinspirados funcionalizados con citosina para la administración ocular de ácido transferúlico. El ácido transferúlico es uno de los antioxidantes naturales más potentes y ÁNGELA VARELA GARCÍA 12 puede eliminar especies reactivas de oxígeno (ROS) y de nitrógeno (RNS), así como regular los sistemas citoprotectores. A nivel ocular, es útil en el tratamiento de lesiones corneales y puede suprimir la producción de amiloide a nivel del cristalino. El principal inconveniente es que su biodisponibilidad oral es inferior al 20%, por lo que resulta conveniente diseñar formulaciones que permitan su administración tópica ocular. La creación de receptores artificiales utilizando la técnica de moldeado molecular (molecular imprinting) no es aplicable cuando la molécula de interés es un antioxidante, ya que se degradaría durante la polimerización y, además, haría que ésta fuese incompleta. Por lo tanto, el desarrollo de hidrogeles con afinidad por antioxidantes requiere la identificación de grupos funcionales que puedan actuar como receptores, sin necesidad de llevar a cabo la polimerización en presencia de la molécula de interés. Muchos fármacos basan su mecanismo de acción en su capacidad para interaccionar con las bases púricas (adenina y guanina) o pirimidínicas (timina, citosina y uracilo), que constituyen el ADN y el ARN. Esta etapa de la Tesis se planteó partiendo de la hipótesis de que la incorporación de una base pirimidínica, como la citosina, a la estructura de un hidrogel debe dotarlo de afinidad por las moléculas con estructura complementaria en términos de capacidad para establecer puentes de hidrogeno e interacciones hidrofóbicas п-п. La utilización de bases nitrogenadas como grupos funcionales no ha sido ensayada previamente, por lo que para su incorporación a los Resumen 13 hidrogeles se puso a punto un procedimiento de anclaje postpolimerización. Para llevar a cabo el estudio, los hidrogeles se prepararon mezclando HEMA con glicidilmetacrilato (GMA) y etilenglicolfenileter metacrilato (EGPEM) en distintas proporciones. El GMA se utilizó como puente para inmovilizar citosina en los hidrogeles, mientras que el EGPEM se incorporó para evaluar la posibilidad de reforzar la interacción entre el ácido transferúlico y la citosina formando complejos de Rebek. Los hidrogeles se prepararon con un espesor de 0.45 mm y la funcionalización con citosina se llevó a cabo por inmersión en una disolución de citosina en agua:dioxano (1:1 vol/vol), a 80 °C durante 24 h. Tras el lavado, la presencia de citosina se confirmó visualmente bajo luz ultravioleta, puesto que es una molécula altamente fluorescente, y también mediante espectroscopía FTIR-ATR y análisis elemental. Los hidrogeles se caracterizaron en cuanto a grado de hinchamiento, transmitancia y propiedades mecánicas, obteniéndose valores situados dentro de los intervalos admitidos para LC hidrofílicas. La incorporación de ácido transferúlico se llevó a cabo sumergiendo discos de hidrogel en una disolución acuosa del fármaco. Los hidrogeles funcionalizados con citosina dieron lugar a una mayor incorporación de agente antioxidante, hasta 2.5 veces superior y, por lo tanto, a coeficientes de reparto entramado/agua (KN/W) más ÁNGELA VARELA GARCÍA 14 elevados que los hidrogeles sin funcionalizar. Los ensayos de cesión in vitro, que se llevaron a cabo en FLS, revelaron que los hidrogeles con mayor KN/W dan lugar a perfiles de cesión más sostenidos. A continuación, los hidrogeles se sometieron a un ensayo de compatibilidad con membrana corioalantoidea de huevos fecundados (ensayo HET-CAM), que es un método alternativo al ensayo in vivo en animales para evaluar el riesgo de irritación ocular. También se evaluó la compatibilidad de los hidrogeles con células epiteliales de la córnea humana (HCEC) mediante la prueba WST-1. Ambos ensayos confirmaron que los hidrogeles son altamente biocompatibles. Los hidrogeles con mejores propiedades de carga y cesión de ácido transferúlico se ensayaron en cuanto a actividad antioxidante, mediante el test ORAC. Los resultados obtenidos indicaron que la incorporación del ácido transferúlico a los hidrogeles no deteriora su actividad antioxidante. Finalmente, se llevó a cabo un estudio de permeabilidad a través de córnea y esclera. Al igual que el ácido transferúlico libre en disolución, el que se cedió a partir de los hidrogeles mostró una elevada tendencia a acumularse en córnea y esclera, y también a pasar al medio receptor. Los resultados obtenidos indican que la utilización de citosina como componente funcional representa una nueva estrategia para dotar a los hidrogeles de afinidad por moléculas que, como el ácido transferúlico, cuentan en su estructura con grupos Resumen 15 aromáticos y otros grupos con capacidad para formar puentes de hidrógeno. En su conjunto, los resultados de esta Tesis Doctoral abren nuevas posibilidades para desarrollar formas de aplicación tópica ocular, tanto líquidas como sólidas, que proporcionen niveles sostenidos de fármaco en córnea y esclera. Introduction ÁNGELA VARELA GARCÍA 24 Figure 1.4: Schematic representation of the layers of the cornea and the cells that conform it. Adapted from Masterton et al. (2018). This is an open access article under the CC BY-NC-ND license. The uvea is located between the retina and the sclera, and is formed by the ciliary body, the choroid and the iris. It is a pigmented vascular layer. The ciliary body covers the anterior sclera. The choroid is the main pigmented vascular tissue of the eyeball. The iris is located between the cornea and the lens, within the aqueous humor, and divides the anterior and posterior chambers with a central aperture, the pupil (Malhotra et al., 2011). The crystalline or lens is made up of long fibre cells, with proteins and water. It is divided into two parts, nucleus and cortex. It is a transparent and flexible tissue, so that light can pass through it without problems and focus on the retina. It can change the curvature 1. Introduction 25 of the surface when necessary, to adjust the focal distance (Petrash 2013; Wang et al., 2019). The vitreous humor represents 80% of the volume of the eyeball and is divided into three parts: the vitreous nucleus, the vitreous base and the vitreous cortex. It is composed of 99% water, together with inorganic salts, sugars, lipids and proteins. Its function, in addition to providing mechanical support, is to maintain homeostasis in neighbouring tissues, provide nutrients, guarantee optical transparency and protect against the entry of cells or macromolecules that may interfere with the normal function of the eye (Monteiro et al., 2015). The retina is a tissue that covers the internal surface of the eyeball. It consists of six classes of neurons: photoreceptors (cones and rods), bipolar cells, horizontal cells, amacrine cells, ganglion cells and Müller's glia, arranged in several parallel layers. Its function is to convert signals coming from outside into nerve impulses, transmitted from the optic nerve to the brain (Willoughby et al., 2010). The optic nerve is part of the central nervous system. It is made up of retinal ganglion cell axons and supporting glial cells. Its function is to transmit electrical signals from the retina to the brain (Chen et al., 2017). The choroid is a vascular layer located at the back of the uvea and consists of five layers: Bruch’s membrane, the choroiocapillaris, two vascular layers (Haller’s and Sattler’s) and the suprachoroidea. It is composed principally of blood vessels. Its main function is to supply ÁNGELA VARELA GARCÍA 26 oxygen and nutrients to the retina, but it can also act as a thermoregulator and modulator of intraocular pressure (IOP) (Nickla et al., 2010). Finally, the sclera is a protective and supportive outer layer of the eye. It occupies the area between the cornea margin and the optic nerve, which constitutes more than 80% of the eye's surface. Its structure varies with age, being thicker during the first few years of life. Then, it stretches and becomes stiffer. Sclera is formed by collagen fibres type I (90%) and type III (5%), which are included in the proteoglycan matrix. Its function is to maintain intraocular pressure and is where extraocular muscles meet. In addition, it is the main carrier tissue of the eye (Malhotra et al., 2011; Coudrillier et al., 2015). 1.2. Eye-defense mechanisms Eye anatomy and physiology make it autonomous to defend itself from external agents (Gaudana et al., 2010). The eye's defense mechanisms can be classified into three types: mechanical, anatomical and immunological (Akpek et al., 2003). There are many factors that, in combination, provide complete protection to the eye, for example, the continuity of corneal and scleral tissue, blinking and tear composition, among others (McClellan 1997). The problem arises when these eye defense mechanisms also act as barriers to drug delivery. The main barriers are categorized as lacrimal film, corneal and non-corneal barriers and blood ocular barriers (Figure 1.5). 1. Introduction 27 Figure 1.5: Schematic representation of the different defense barriers that the eye presents to the administration of drugs. Adapted from Huang et al. (2018) with permission from Elsevier. 1.2.1. Lacrimal film It is the first protective barrier of the eye. It consists of proteins, lipids and mucin, with perfect electrolyte composition and optimal nutrient and pH values. The lacrimal fluid is made up of a lipid layer, an aqueous layer and a mucous membrane, from the outside to the inside. Its function is to modulate cell migration and proliferation during healing, as well as to modulate normal cell differentiation and ÁNGELA VARELA GARCÍA 28 the secretion of electrolytes and water. The flow of tears is constant, being renewed every 2-20 minutes, so the residence time of any drug on the ocular surface is very low, which reduces its possibility of absorption (Barar et al., 2009; Huang et al., 2018). 1.2.2. Corneal barrier The cornea, with a thickness of 0.5 mm and formed mainly by collagen, is the main barrier to the penetration of molecules into the eye (Sánchez-López et al., 2017; Huang et al., 2018). It consists of five layers, each of which has different properties. The outermost layer is the epithelium (stratified, squamous and not keratinized), which may allow the pass of small hydrophobic molecules but hinders the entrance of hydrophilic drugs. Only molecules with a molecular weight below 500 Da can pass through the paracellular pathway (Barar et al., 2009; Huang et al., 2018). The stroma (intermediate layer) opposes to the passage of lipophilic molecules due to its high content in hydrated collagen and proteoglycans. The endothelium is the innermost layer and consists of a monolayer of polygonal cells. Like the epithelium, it prevents the passage of hydrophilic molecules to the aqueous humor but allows the passage of small lipophilic molecules (Barar et al., 2008; Huang et al., 2018). In the cornea, collagen fibers are present in the Bowman's layer and stroma. In the first one, the fibrils have a diameter between 20-25 nm and their distribution is random, forming leaves 8-12 µm thick. In the stroma, the diameter of collagen fibers varies between 25-35 nm and its 1. Introduction 29 distribution is in parallel, forming flat lamellar bundles (Komai et al., 1991). The physicochemical properties of drugs also affect their penetration through the cornea. Adequate balance between lipophilia, molecular weight and degree of ionization is required for a successful passive diffusion. The most common way of passage of drugs through the cornea is transcellular, while the paracellular predominates for hydrophilic or low molecular weight drugs (Barar et al., 2009; Sánchez-López et al., 2017). 1.2.3. Non-corneal barriers The conjunctiva is a thin mucous membrane that covers the eyelids internally and the anterior surface of the sclera. Being formed by blood capillaries and conjunctival lymphatics, productive absorption of a drug is very low, although the permeability is greater than in cornea and also allows the passage of hydrophilic molecules and molecular weights up to 10 kDa (Sánchez-López 2017; Huang et al., 2018). The sclera consists mainly of collagen and mucopolysaccharide. Collagen fibers vary in size from 25 to 230 nm. Although they form lamellar bundles, individual fibrils are more randomly arranged than in the cornea. Thickness varies from 0.5-6 µm. In the outermost part of the sclera, the collagen bundles are narrower and thinner than in the inner part (Komai et al., 1991). Sclera has a surface area of about 16 cm2 and it is more permeable to hydrophilic solutes than other ocular structures, such as the cornea, as they can diffuse through the aqueous ÁNGELA VARELA GARCÍA 30 medium between collagen fibrils, rather than through cell membranes. In this case, the radius of the molecules better predicts permeability than molecular weight (MW). Thus, molecules with higher MW but smaller radius cross the sclera better than others of the same MW but larger radius. Molecular charge also influences the transport of molecules; negatively charged molecules are more permeable than those with positive charge, due to the charge of the proteoglycan matrix, which is negative. Overall, drug permeability through sclera is favored compared to cornea (Barar et al., 2008; Huang et al., 2018). 1.2.4. Blood ocular barriers There exist two types of ocular barriers in charge of regulating the solutes that cross towards the internal zones of the ocular globe: the haemato-aqueous barrier (BAB) and the haemato-retinal barrier (BRB). The BAB is located at the anterior region and consists of the endothelium of the iris/ciliary blood vessels and the non-pigmented ciliary epithelium. Its function is to regulate the intraocular pressure, turning the flow of aqueous humor, to maintain the transparency and chemical composition of the ocular fluids. It also regulates the passage of drugs from the anterior to the posterior segment. The BRB is located at the posterior region of the eye and also consists of two types of cells: capillary endothelial cells of the retina and cells of the retinal pigment epithelium, which form the internal and external BRB, respectively. Its main function is to hinder the diffusion of substances 1. Introduction 31 from the circulatory torrent to the retina. The main limiting property for the passage of substances is the molecular radius. Thus, the permeability decreases as the radius increases. Lipophilia also influences; therefore, only small and lipophilic molecules can pass from the choroid to the retina (Huang et al., 2018). 1.3. Ocular drug administration Drug administration at the anterior segment of the eye can be done in several forms. Topical administration is the most common route for the treatment of diseases of the anterior segment, usually as eye drops or ointments. The following advantages can be mentioned: it is a non-invasive and painless pathway with high patient compliance; very high doses of the drug are not needed; and the effect may be immediate. The main disadvantage is that ocular bioavailability of drugs administered topically is less than 5% due to both physicochemical barriers (the structure of the eye or the composition of the tear) as well as naso-lacrimal drainage, tearing or blinking reflexes or the low volume that can host the cul-de-sac. In addition, it is estimated that the tear volume is 7 μL, and the restoration time of the tear film is fast (2-3 minutes), so that topically administered solutions would be eliminated shortly after instillation (Awwad et al., 2017; Djebli et al., 2017). Moreover, systemic side effects may arise, as much of the instilled dose passes into the bloodstream through the conjunctiva and nasal mucosa. In addition, nasolacrimal drainage of certain substances can cause toxic reactions ÁNGELA VARELA GARCÍA 32 (Loftsson et al., 1999; Ribeiro et al., 2015). This system connects the flow of tears from the eye to the nasal cavity. The part of the drug drained after topical application passes into the lacrimal sac and then into the nasolacrimal duct until it reaches the nose. During this passage, the drug passes through vascularized areas, where it can be absorbed into the systemic circulation, which can lead to undesirable side effects (Bachu et al., 2018). Ophthalmic preparations must meet some specific requirements. The active molecule must have a certain aqueous solubility. In addition, there are several critical parameters that need to be monitored. The ophthalmic formulations must have a tolerable acidity, with an adequate pH around 7.4, although there are some exceptions. They must also be isotonic as well as stable at room temperature. Other critical factors to consider are drug pKa and formulation viscosity. Excipients used during manufacture must be free from toxicity and should not interact with the packaging. All ophthalmic products must be sterile, and injectables must also be free of endotoxins and particles (Novack et al., 2016; Yellepeddi et al., 2016) (Figure 1.6). Drugs can be administered to the anterior segment of the eye also though intracameral and subconjunctival injections. Both avoid the cornea and hematoencephalic barrier and provide high ocular bioavailabilities. Ocular injections present all the inconveniences associated to injectable formulations, aggravated by the sensitive region where the formulations are delivered, which 1. Introduction 33 causes patient discomfort and has the risk of tissue damage and infection. Finally, the systemic oral route despite being a non-invasive is barely used, since very high doses of drug are necessary to exert an effect at the ocular level and there are many adverse effects on other tissues (Janagam et al., 2017). Figure 1.6. Penetration and elimination pathways of drug after topical administration. Adapted from Janagam et al. (2017) with permission from Elsevier. The administration of drugs to the posterior segment of the eye is more complicated. Topical forms, such as drops or ointments, can hardly reach the posterior segment, except if the drug can efficiently enter through the conjunctiva-sclera pathway. Drugs can be administered orally, intramuscularly or intravenously, but the vast majority of drugs cannot cross the barriers, so bioavailability is ÁNGELA VARELA GARCÍA 40 worldwide, and this figure is expected to double in 30 years (Tan et al., 2019). Reactive oxygen species (ROS) are a subproduct of normal anaerobic metabolism. Enzymes such as SOD (superoxide dismutase), CAT (catalase) or GPx (glutathione peroxidase) neutralize these subproducts. When a disequilibrium is generated in the redox haemostasis of the pro- and antioxidant systems due to the incapacity of enzymes to eliminate free radicals, cell necrosis is triggered due to damage to proteins, lipids and DNA, causing degeneration at the ocular level (Bungau et al., 2019; Tan et al., 2019). There are different factors that promote oxidative stress, such as age, exposure to light or metabolic processes, such as hyperglycemia (Bungau et al., 2019). The cornea protects the eye from environmental stress by absorbing ultraviolet (UV) light, but prolonged irradiation may cause corneal lesions (Zernii et al., 2018). The radiation can end up damaging the anterior segment. The most common acute condition is photokeratitis and in the long-term cataracts, carcinomas, melanomas or other pathologies of the conjunctiva may appear. The most serious consequence of UV radiation is ROS generation. At the ocular level, there are several antioxidants of low molecular weight in both tissues and fluids, such as ascorbic acid, alpha-tocopherol or glutathione, and of high molecular weight, which are the enzymes mentioned above. The cornea, especially the anterior part, can absorb up to 60% of UVA radiation and up to 92% of UVB radiation, 1. Introduction 41 although it is more sensitive to the latter. Although the eye has an antioxidant defense, when there is an increase in UV radiation, there is a prooxidant/antioxidant imbalance that favors the damage (Cejkova et al., 2004). Several carotenoids and polyphenols have antioxidant and antiinflammatory activities. They decrease the production of ROS, which in turn inhibits tumor necrosis factor α (TNFα) and vascular endothelial growth factor pathways, which helps to suppress p53- dependent apoptosis, eliminating the genesis of inflammatory markers (IL-8, 6, 1a and endothelial leucocyte adhesion molecule-1) (Bungau et al., 2019). One example is resveratrol, which has also been shown to exhibit anti-aging properties at ocular level (Abu-Amero et al., 2016). Another compound to highlight is transferulic acid, which besides being a powerful antioxidant, exerts anti-inflammatory and antibacterial effects, among others. Its mechanism of action, in addition to eliminating free radicals, can inhibit the enzymes that catalyze the synthesis of these free radicals (Zdunska et al., 2018). At the ocular level, studies have shown that it can be useful for the healing of corneal wounds (Tsai et al., 2016; Grimaudo et al., 2020), or to suppress the production of amyloid B in the human lens (Nagai et al., 2017). 1.5. Ocular viral infections It is estimated that 20-70% of conjunctivitis are viral, and of these, 65-90% are caused by adenoviruses. These viral infections ÁNGELA VARELA GARCÍA 42 spread through the air, by deposits, or by direct contact with the virus. Conjunctivitis can be classified in two groups: papillary or follicular. The first one manifests itself with flat and compact projections, with numerous eosinophils, lymphocytes, plasma cells and mast cells in the stroma that surrounds a central vascular channel. It is usually associated with a foreign body response or an allergic immune response. Follicular conjunctivitis occurs with prominent follicles in the lower palpebral and forniceal conjunctiva. The most common symptoms and signs during viral conjunctivitis are foreign body sensation, red eyes, itching, sensitivity to light, burning, and watery discharge (Li et al., 2018; Solano et al., 2019). Keratitis is another eye infection caused by a virus; in this case mainly herpes simplex virus (HSV). The number of people affected by this disease worldwide was estimated at 1.5 million. It is the most common cause of unilateral infectious corneal blindness. Unlike bacterial or fungal keratitis, viral keratitis can be recurrent and chronic. There are two other forms of viral keratitis, but they are less common: varicella-zoster virus (VZV) keratitis and cytomegalovirus (CMV) keratitis (Austin et al., 2017). The classic symptoms of viral keratitis are eye pain, blurred vision, redness, and photophobia (Rowe et al., 2013). Herpes zoster is a relevant eye infection. It has its origin in the reactivation of the varicella-zoster virus (VZV), a virus of the herpesviridae family (Table 1.1). Initially, the disease presents itself 1. Introduction 43 as chickenpox, infecting the sensory ganglia. It usually occurs in the early stages of life. It is very contagious but benign and occurs in the form of blisters spread throughout the body with itching. Table 1.1. Classification of Herpesvirus. Herpesviridae family Sub-family Types Virus Alpha-herpesvirinae Herpesvirus 1 Herpes simplex 1 Herpesvirus 2 Herpes simplex 2 Herpesvirus 3 Varicella Zoster Beta-herpesvirinae Herpesvirus 5 Cytomegalovirus Herpesvirus 6 Herpesvirus lymphotrope Herpesvirus 7 Human herpesvirus Gamma-herpesvirinae Herpesvirus 4 Epstein-Barr Virus Herpesvirus 8 Kaposi's Sarcoma The reactivation of the virus, known as herpes zoster or shingles, is more dangerous. It manifests as vesicular eruptions, affecting mostly the thoracic nerve, but also the trigeminal nerve. Factors such as changes in T-cells or a decrease in the neutralization of antibodies, which occur as age progresses, influence the possible reactivation of VZV. The risk is also higher in immunocompromised patients. The data warn that about 50% of adults affected by herpes zoster are at risk of complications. Herpes zoster ophthalmicus represents between 10- 20% of cases of zoster and is the second most common complication. The disease begins with symptoms of severe pain and discomfort. ÁNGELA VARELA GARCÍA 44 After a few days, skin lesions appear. Complications at the level of the cornea appear in 65% of cases. The acute phase presents with epithelial keratitis (puncture or dendritic) or stromal keratitis, and the late phase with neurotrophic keratopathy or neovascularization of the cornea. The most common manifestations are photosensitivity, decreased vision or perforation, among others (Zhu et al., 2014). 1.5.1. Treatment of ocular viral infections The causal agent of viral conjunctivitis should be identified correctly so that proper treatment can be given. For example, commonly viral conjunctivitis occurs with watery discharge, as opposed to bacterial conjunctivitis that presents mucopurulent discharges. There is no effective treatment in its entirety. Artificial tears, topical antihistamines, or cold compresses are used as palliatives. The effectiveness of ocular antivirals is not high. Topical antibiotics are not indicated since they are not capable of preventing secondary infections, also causing undesired side effects, such as allergy or toxicity. Of all cases of acute conjunctivitis, the herpes simplex virus is responsible for 1.3-4.8%. This type of pathology is usually unilateral, with watery discharge and sometimes even vesicular lesions may appear on the eyelid. The usual treatment consists of topical and oral antivirals. The use of topical corticosteroids should be avoided because they are potentiators of the virus (Azari et al., 2013). 1. Introduction 45 On the other hand, topical treatment for viral keratitis caused by herpes simplex virus (HSV) includes antiviral drugs and adjunctive topical corticosteroids. The most commonly used topical antiviral was trifluridine, although its bioavailability was very low and caused surface toxicity at the ocular level, so its use has been reduced. Acyclovir is the first choice for the treatment of keratitis since its effectiveness is high and it is less toxic. New synthetic drugs, such as ganciclovir, have a broader-spectrum antiviral action, i.e., in addition to treating keratitis caused by HSV, it also attacks VZV and CMV. Additionally, topical corticosteroids are sometimes used as adjuvant therapy. On the other hand, acyclovir is also given as an oral adjuvant therapy. Valacyclovir was also seen to have greater oral bioavailability for the treatment of HSV, requiring lower doses (Austin et al., 2017). Finally, herpes zoster ophthalmicus usually appears as a mild case, but antiviral use is still recommended for the first 72 hours after the onset of the rash. Early treatment can lead to a reduction in the duration of the illness as well as acute pain and halve the likelihood of eye disorders. The drugs chosen are acyclovir, valacyclovir and famcyclovir. Although all three have similar efficacies, the more recent ones (valacyclovir and famcyclovir) have higher oral bioavailability. In this case, corticosteroids are also used as adjuvant therapy, especially during the first stage of the disease, to relieve pain and improve healing, but their long-term use should be avoided ÁNGELA VARELA GARCÍA 46 because of side effects. If the disease manifests late, antiviral drugs are not effective and topical corticosteroids should be used to reduce inflammation (Zhu et al., 2014). 1.6. Micelles and contact lenses as platforms for ocular delivery The classic methods of ocular administration of drugs (eye drops or ointments) have different drawbacks, as explained in section 1.3. That is the reason of a continuous search for new drug dosage forms, to improve drug ocular bioavailability, combining an efficient controlled release and minimizing pain and discomfort for the patient. Among other platforms, polymeric micelles and soft contact lenses are gaining increasing interest (Gote et al., 2019). 1.6.1. Polymeric micelles The use of nanocarriers is gaining increasing attention for the delivery of ocular drugs (Gomez-Ballesteros et al., 2019). Polymeric micelles are formed by amphiphilic copolymers, which spontaneously self-assemble in an aqueous medium, once they exceed the critical micellar concentration (CMC) (Grimaudo et al., 2019). Their size can vary between 5-100 nm, and their shapes can also be different. During the process of micelle formation there is an equilibrium of intermolecular forces, such as van der Waals forces, hydrogen bonding and hydrophobic, steric and electrostatic interactions (Gaucher et al., 2005). The structure of the micelle comprises an internal hydrophobic core, capable of hosting liposoluble drugs and solubilizing them, and an external hydrophilic shell, in contact with 1. Introduction 47 the external environment, which physically stabilizes the micelle (Figure 1.7) (Croy et al., 2006). Figure 1.7. Schematic representation of the structure of a polymeric micelle. Figure made by the author of this Thesis. The encapsulation of drugs in polymeric micelles prevents the interaction with the surrounding environment, which enhances their physicochemical stability. In addition, properties of the external shell, such as viscosity, thickness or porosity, may determine the rate of drug release. The process of drug incorporation is complex and depends on the molecular and physicochemical properties of both parts (micelle and drug). The molecular weight and the hydrophiliclipophilic balance (HLB) are properties to consider in the copolymer; for similar molecular weights, a decrease in HLB leads to larger nucleus and with greater encapsulation power. On the other hand, for similar HLB, the higher the molecular weight of the copolymer, the more efficient the encapsulation is. Properties of the drug such as molecular weight, radius, lipophilia (partition coefficient, logP), ÁNGELA VARELA GARCÍA 48 melting point, tendency to aggregation and the presence of specific functional groups that may interact with the micelle modify the encapsulation efficiency. There are different methods for the preparation of drug-loaded polymeric micelles. The most direct and simple method consists of dissolving the copolymer in water, stabilizing the micelle dispersion at a suitable temperature (48-72 h) and then adding the drug to be incorporated into the micelles. This method is generally used for micelles formed by copolymers of intermediate HLB. It may require the application of heat to dehydrate the segments that will form the nucleus to form the micelles. Another technique, used for more hydrophobic copolymers, consists of dissolving both components (copolymer and drug) in water-miscible organic solvents. The mechanism of micellar formation will depend on the procedure by which the organic solvent is removed. For example, the mixture may be dialyzed and the slow elimination of the organic phase triggers the formation of micelles; or the organic phase may evaporate, forming a polymeric film, which will be rehydrated with an aqueous solvent, aided by heat, to form the drug-loaded micelles. There are more techniques, such as trapping a hydrophobic drug in an O/W emulsion, casting in solution or lyophilization (Gaucher et al., 2005). Topical formulations of drug-loaded polymeric micelles can be considered as non-invasive delivery systems. These systems can stay in the site of administration long enough for the drug to exert its 1. Introduction 49 therapeutic effect due to properties such as viscosity or mucoadhesion. In general, the contact time of the formulation is directly proportional to its viscosity. The clearance of drugs by blinking or naso-lacrimal drainage is reduced by increasing the viscosity of the system. The use of mucoadhesive components for the formation of the polymer micelles also increases the residence time of the micelles at the administration site due to the formation of covalent bonds with mucin. Polymeric micelles are safe and effective systems and avoid patient discomfort. For the drug to reach the posterior segment of the eye, it can follow the corneal or conjunctival-scleral route (Figure 1.8) (Mandal et al., 2017; Grimaudo et al., 2019). The size of the polymeric micelles in the nanoscale endows them with the ability to pass through structures such as the cornea or the conjunctiva/sclera pathway. The pass of hydrophobic drugs through the hydrophilic stroma may be favored by the encapsulation in nanomicelles. The sclera has a larger area, which allows the polymeric micelles to spread laterally to the posterior segment. From there, the cells of the retinal pigmented epithelium may engulf nanocarriers by endocytosis, opening the possibility of exerting an effect on the ocular tissues of the posterior segment (Hughes et al., 2005; Mandal et al., 2017). ÁNGELA VARELA GARCÍA 56 rate. Critical parameters such as the selection of the functional monomer and its stoichiometry with respect to the template must be considered (Alvarez-Lorenzo et al., 2010). g) Supercritical fluid impregnation: Supercritical fluids, mainly supercritical CO2 (scCO2) can be used to enhanced drug dissolution and penetration into polymer networks (Garcia- Gonzalez et al., 2015). The process starts with the dissolution of the drug in the supercritical solvent in contact with the CLs. This technique allows the loading of hydrophilic and hydrophobic drugs at higher amount than conventional soaking in aqueous medium, but no significant improvements in the control of release rate are usually achieved (Yañez et al., 2011). h) Incorporation of colloidal nanoparticles: the administration of drugs encapsulated in colloidal nanoparticles increases the residence time of the drug in the cornea and prevents the ocular enzymes from metabolizing the drug. Therefore, the addition of nanocarriers to CLs may prolong the action time. Nevertheless, the addition of nanoparticles may obstruct the vision by decreasing the transparency of the lens. There are four methods of preparing these systems. The first one consists of preparing the nanoparticles loaded with drug, and their subsequent dispersion in pre-monomer mixtures, to 1. Introduction 57 form CLs. The second method is to add surfactants and drugs to pre-monomer mixtures to form micelles during polymerization. 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Aims The eye is a peculiar organ, protected by a combination of anatomical, mechanical and immunological barriers. Although it is easily accessible for topical drug administration, the combination of local and systemic barriers prevents a successful drug distribution. Formulations that can combine the patient acceptance of topical formulations and the effectiveness of intraocular injections have been largely prospected. The aim of this PhD Thesis was to design polymeric micelles and CL with improved features for topical administration of ophthalmic drugs. Specifically, the research focused on the antiviral drugs acyclovir and valacyclovir and the antioxidant agent transferulic acid. These drugs are poorly soluble in water and their therapeutic outcomes could be notably improved if sustained levels on the eye structures are achieved. According to this general aim, three specific aims were identified and developed as follows. 1) Design of polymeric micelles suitable for the administration of acyclovir. This antiviral agent is the first choice for the treatment of ÁNGELA VARELA GARCÍA 74 ocular herpes caused by the herpes simplex virus. This virus can affect all layers of the cornea, producing blurred vision, redness, or tearing. Herpes simplex keratitis (infection and inflammation of the cornea) is the leading cause of infection blindness worldwide. Acyclovir penetrates infected cells and competes with natural nucleosides to incorporate DNA and act as a chain terminator. After anchoring, a suicidal inactivation mechanism occurs, since the terminated DNA binds to the viral DNA polymerase and irreversibly inhibits it, preventing viral replication. Classic topical treatment requires a long application period and results in serious side effects. Oral administration is characterized by having a bioavailability of less than 20%, which is the reason of prescribing high doses for long periods of time. The hypothesis of this first stage of the Thesis was that the encapsulation of acyclovir in polymeric nanomicelles should increase drug solubility of the drug and promote the accumulation of the drug in cornea and sclera and its access to deeper structures. Soluplus and Solutol copolymers were chosen as amphiphilic copolymers. Soluplus is a biodegradable copolymer of polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol (MW 90000-140000 g/mol, CMC 7.6 mg/L), which forms aqueous dispersions that may undergo in situ gelling at the ocular temperature. This additional feature may increase the residence time at the site of application and enhance the control of drug release. Solutol or macrogol 15-hydroxystearate (MW 963.24 2. Aims 75 g/mol, CMC 0.005-0.02%) is a non-ionic surfactant, which improves stability and solubility of insoluble drugs. It is stable, highly biocompatible and permeable to the mucosa. To carry out the work, dispersions of each copolymer will be prepared covering a wide range of concentrations and their size, Z potential, capability to host acyclovir and rheological behaviour will be evaluated. Those formulations combining adequate performances will be tested regarding cornea and sclera accumulation and permeability. 2) Design of imprinted hydrogels for antiviral drugs. Seeking for prolonging drug permanence on the ocular surface beyond that the formulations in nanomicelles may allow, the aim of the second step of the Thesis was to design hydrogels suitable for soft CL with affinity for acyclovir and valacyclovir. Among the proposed procedures to endow the hydrogel CLs with affinity for specific molecules, the creation of artificial receptors using the molecular imprinting technique stands out. This technique requires incorporating the substances of interest into the monomers mixture so that these can rearrange according to their affinity. This rearrangement becomes permanent during polymerization. The removal of the template molecules generates cavities with the most appropriate size and chemical groups to host the substance of interest again. Functional monomers suitable for interaction with the antiviral drugs will be first screened using computational modeling. Then hydrogels will be ÁNGELA VARELA GARCÍA 76 prepared with various contents in the functional monomer in the presence (imprinted) and absence (non-imprinted) of the drug. The load and release capacities of antiviral drugs will be evaluated, as well as their biocompatibility in chorioallantoic membrane (HET-CAM test). The hydrogels will be characterized in terms of swelling, transmittance and mechanical properties. Finally, the permeability through bovine cornea and sclera of the aqueous solution of the drug and the drug released by the hydrogels will be compared. 3) Design of cytosine-functionalized bioinspired hydrogels for ocular administration of transferulic acid. Transferulic acid is one of the most powerful natural antioxidants, it scavenges reactive oxygen and nitrogen species (ROS and RNS), and it regulates cytoprotective systems. At the ocular level, it is useful in the treatment of corneal lesions and can supress the production of amyloid at the crystalline. However ocular formulations for sustained release of transferulic acid have not been developed yet. The creation of artificial receptors using the molecular imprinting technique is not applicable when the molecule of interest is an antioxidant since it would degrade during polymerization and would also interfere with the process. Therefore, the development of hydrogels with an affinity for antioxidants requires the identification of functional groups that can act as receptors without the need of carrying out the polymerization in the presence of the molecule of interest. The mechanism of action of 2. Aims 77 several drugs relies on their ability to interact with puric bases (adenine and guanine) or pyrimidine bases (thymine, cytosine and uracil) that build up DNA and RNA. The hypothesis of the last part of the Thesis was that the incorporation of a pyrimidine base, such as cytosine, into the structure of a hydrogel should endow it with an affinity for molecules with complementary structure in terms of ability to interact through hydrogen bonding and hydrophobic п-п stacking. The use of nitrogenous base as functional groups has not been previously tested, so a post-polymerization anchoring procedure was developed for their incorporation into the hydrogels. To carry out the study, the hydrogels will be prepared by mixing HEMA with different proportions of glycidylmethacrylate (GMA) and ethylene glycolphenyl methacrylate (EGPEM). GMA can serve as a bridge to immobilize cytosine in hydrogels, while EGPEM may reinforce the interaction between transferulic acid and cytosine forming Rebek molecular complexes. The hydrogels will be functionalized with cytosine and characterized regarding the amount grafted and in terms of degree of swelling, transmittance and mechanical properties. The capability of the hydrogels to load transferulic acid and to sustain its release while preserving the antioxidant activity will be evaluated in detail. HET-CAM test and viability of human corneal epithelial cells (HCEC) will be used for a first assessment of the ocular compatibility of the developed hydrogels. Finally, permeability of transferulic acid ÁNGELA VARELA GARCÍA 78 through cornea and sclera when applied in solution and when delivered from the hydrogels will be compared. Polymeric micelles for acyclovir ocular delivery: formulation and cornea and sclera permeability 3. Polymeric micelles for acyclovir ocular delivery: formulations and cornea and sclera permeability 81 3. Polymeric micelles for acyclovir ocular delivery: formulation and cornea and sclera permeability 3.1. Introduction The eye is considered a unique tissue from an immunological perspective. Several protective elements are present on the ocular surface; for example, mucins that form a dense glycocalix on the cornea provide a physical barrier that prevents bacterial adhesion, and β-defensins, calprotectin, lysozyme, lipocalin and lactoferrin exhibit antimicrobial features. Eye surface infections appear when the homeostasis is disturbed by both unfavorable environmental conditions and infectious agents, which break down the eye surface and alter the innate immune system of this organ (Caspi, 2013; Pearlman et al., 2013; Lu et al., 2016). Ocular infections can be caused by bacteria and fungi pathogens (Caspi, 2013; Mangoni et al., 2016) or by viruses such as Herpes simplex, Varicella Zoster or Cytomegalovirus (Edwards et al., 2017). The varicella zoster virus (VZV) causes chickenpox and herpes zoster. The first is a benign disease and the second appears after a ÁNGELA VARELA GARCÍA 88 1000N (TA Instruments, UK) rheometer fitted with a Peltier plate and a 6-cm in diameter cone (2 º). The angular frequency was fixed at 5 rad/s and the oscillation stress at 0.1 Pa. 3.2.4. Acyclovir solubilization Soluplus and Solutol solutions (3 mL) were poured in 5 mL Eppendorf tubes containing acyclovir in excess (50 mg). Solubility of acyclovir in water and PBS pH 7.4 was also recorded. The systems were maintained under magnetic stirring for 72 h, at 300 rpm and room temperature. Then, they were centrifuged at 5000 rpm for 30 min to separate non-solubilized acyclovir. Absorbance of the supernatants was measured at 252 nm (UV/Vis spectrophotometer Agilent 8453, Germany) previous dilution with water:ethanol 50:50 v/v mixtures. Acyclovir concentration was calculated using a calibration curve previously prepared. The solubility data was used to calculate the following parameters (Alvarez-Rivera et al., 2016). Molar solubilization capacity (moles of drug that can be solubilized per mol of copolymer forming micelles): 𝜒=    [Eq. 3.1] Micelle-water partition coefficient (ratio between the drug concentration in the micelle and the aqueous phase): 𝑃 =   [Eq. 3.2] 3. Polymeric micelles for acyclovir ocular delivery: formulations and cornea and sclera permeability 89 Molar micelle-water partition coefficient (which eliminates the P dependence on the copolymer concentration, assigning a default concentration of 1M): 𝑃𝑀 =∗( )  [Eq. 3.3] Gibbs standard-free energy of solubilization was estimated from the molar micelle/water partition coefficient (PM) and the micelle-water partition coefficient (P), as follows 𝛥𝐺𝑠 = −𝑅𝑇∗ln(𝑃𝑀) [Eq. 3.4] 𝛥𝐺𝑠 = −𝑅𝑇∗ln(𝑃) [Eq. 3.5] In these equations, Stot represents the total solubility of acyclovir in the micellar solution, Sw is the acyclovir solubility in water, CMC is the critical micelle concentration, Ccopol is the copolymer concentration in each micelle solution, and R is the universal constant of gases. 3.2.5. Micelle stability against dilution Aliquots of acyclovir-loaded Soluplus (12 %w/w) micelle dispersions prepared in PBS pH 7.4 were poured onto temperaturecontrolled (35 ºC) quartz cells already containing PBS, so the dispersions were diluted 30- or 60-fold. The absorbance of the samples was immediately measured at 252 nm and each 30 seconds during 30 min (UV/Vis spectrophotometer Agilent 8453, Germany). Similar experiments were also carried out in triplicate using water and SLF as dilution medium. ÁNGELA VARELA GARCÍA 90 3.2.6. Corneal and sclera permeability assay Acyclovir permeability assays through cornea and sclera were carried out following the BCOP test protocol (Alvarez-Rivera et al., 2016; OECD, 2017; Alvarez-Rivera et al., 2018). Bovine eyes were collected in the first hour after dead, from a local slaughterhouse. They were transported completely immersed in PBS solution with antibiotics added (penicillin 100 IU/mL and streptomycin 100 μg/mL) and maintained in an ice bath. Next, corneas were isolated with 2-3 mm of surrounding sclera, or scleras were isolated. In both cases, tissues were washed with PBS, before mounted on vertical diffusion Franz cells. Donor and receptor chambers were filled with carbonate buffer pH 7.2. The receptor chambers were placed inside a bath at 37 ºC and kept under magnetic stirring during 1 h in order to balance ocular tissues. Then, the volume of the donor chamber was completely removed and replaced by drug solutions (2 mL). Acyclovir aqueous solution (0.3 mg/mL) was prepared dissolving the required amount in water. Saturated acyclovir solution in Soluplus micelles (20% w/w) was prepared as reported above; the final drug concentration was 1.34 mg/mL. The chambers were covered with parafilm to prevent evaporation (0.785 cm2 area available for permeation). Samples (1 mL) were removed from the receptor chamber at 0.5, 1, 2, 3, 4, 5 and 6 h, replacing the same volume with carbonate buffer each time, and taking care of removing bubbles from the diffusion cells. All experiments were carried out in triplicate. 3. Polymeric micelles for acyclovir ocular delivery: formulations and cornea and sclera permeability 91 Acyclovir permeated was quantified by HPLC (Waters 717 Autosampler, Waters 600 Controller, 996 Photodiode Array Detector), fitted with a C18 column (Waters Symmetry C18, 5 μm, 4.6×250 mm) and operated using Empower2 software. Mobile phase was water:acetonitrile (95:5) at 1 mL/min and 35 ºC. The injection volume was 50 µL and acyclovir was quantified at 251 nm (retention time 4.5 min). Standard solutions were 0.075-15 µg/mL of acyclovir in water (Volpato et al., 1997). The cumulative amounts of acyclovir permeated per area versus time were fitted to a linear regression, and the steady state flux (J) and the lag time (tlag) were obtained from the slope and the x-intercept of the linear regression, respectively (Al- Ghabeish et al., 2015). After 6 h permeation test, aliquots of the donor chambers were taken for subsequent analysis. Corneas/scleras were immersed during 24 h in 3 mL of ethanol:water (50:50 v/v) medium, sonicated during 99 min at 37 ºC, and centrifuged (1000 rpm, 5 min, 25 ºC), and the supernatant filtered (Acrodisc® Syringe Filter, 0.22 µm GHP Minispike, Waters), centrifuged again (14000 rpm, 20 min, 25 ºC) and filtered to be measured in HPLC (Volpato et al., 1997). 3.3. Results and Discussion 3.3.1. Micelles preparation and characterization Soluplus and Solutol micelles were prepared in water and PBS pH 7.4 using copolymer concentrations up to 20% w/w. The pH of the Soluplus solutions in water was acid (pH 3.2) while Solutol solutions ÁNGELA VARELA GARCÍA 92 were nearly neutral (pH 6.5). The different pH between copolymer solutions could affect to the acyclovir solubility (pKa 2.27 and 9.25). CMCs of Soluplus and Solutol were reported to be 6.6x10-5 mM and 5.19x10-2 mM, respectively (BASF, 2010; BASF, 2012). The concentrations of each copolymer chosen for the study were well above the CMC values, and ranged between 0.09-1.74 mM for Soluplus, and 10.38-207.63 mM for Solutol (corresponding to 1-20% copolymer concentration). Size and Z-potential of micelles prepared with 12% copolymer are shown in Table 3.1. Soluplus micelles had an average size of 117.4 nm and a polydispersity index of 0.23, while Solutol micelles were much smaller showing an average size of 18.7 nm and a polydispersity index of 0.18. These values are in good agreement with literature (Hou et al., 2016). Loading of acyclovir caused a minor increase in the size of Soluplus micelles (137.0 nm), but remarkably increased the size of Solutol ones (134.9 nm). The surface charge of both types of copolymer was similar, being slightly negative or close to zero. 3. Polymeric micelles for acyclovir ocular delivery: formulations and cornea and sclera permeability 93 Table 3.1. Size, polydispersion index (PDI) and Z-potential of Soluplus and Solutol (12%) micelles in PBS pH 7.4 before and after being loaded with acyclovir (ACV). Mean values and standard deviations in parenthesis; n=3. Formulations Particle size (nm) PDI Z-potential (mV) Soluplus 117.4 (1.4) 0.23 (0.01) -1.73 (0.94) Solutol 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) Solutol + ACV 134.9 (1.4) 0.26 (0.02) -1.93 (0.54) 3.3.2. Rheological behavior Solutol (12 and 20%) dispersions showed viscous-like behavior with negligible G’ values in the 15 to 40 ºC interval (Figure 3.2). The viscosity slightly decreased with the increase in temperature. The complex viscosity values recorded were in the range 0.004 to 0.007 Pa.s, which is slightly higher than pure water. Differently, Soluplus dispersions showed a remarkable increase in the values of both G’ and G’’ when temperature surpasses 30 ºC or 27 ºC in the case of 12 and 20% dispersions, respectively (Figure 3.2). The increase was almost linear in the 30 to 37 ºC range, and the G’’ values at 37 ºC were close to 7-40 Pa (the highest values were recorded in PBS) and 1500 Pa for 12 and 20% dispersions, respectively. This behavior agrees well with previous reports on Soluplus dispersions (Alvarez-Rivera et al., 2016) and it is quite different from that showed by common in situ gelling copolymer ÁNGELA VARELA GARCÍA 94 dispersions that exhibit a sharp increase in G’ and G’’ at the gelling temperature. The complex viscosity values recorded at 35 ºC for Soluplus 12% in water and in PBS were 0.50 and 3.12 Pa.s, respectively, and for Soluplus 20% in water and in PBS were 103.4, and 79.7 Pa.s. These values suggest that the micelle formulations could remain for prolonged time on the ocular surface. 3.3.3. Acyclovir solubilization Solubility of acyclovir in water was 1.02 mg/mL, a value slightly lower than the values previously reported in literature (Majumdar et al., 2009). Using slightly alkaline medium, such as PBS pH 7.4 or SLF pH 7.5, the solubility increased to 1.44 and 1.56 mg/mL, respectively. This part of the study was aimed to elucidate whether Soluplus and Solutol micelles could encapsulate acyclovir, increasing its apparent solubility. In the range of copolymer concentrations tested, acyclovir solubility positively correlated with Soluplus concentration. The enhancement in solubility was slightly higher in PBS than in water, with apparent solubility values of 2.10 (s.d. 0.07) and 2.05 (s.d. 0.04) mg/mL, respectively, in 20% w/w Soluplus micelle medium. 3. Polymeric micelles for acyclovir ocular delivery: formulations and cornea and sclera permeability 95 Figure 3.2. Effect of temperature on G’ and G’’ values of Solutol and Soluplus dispersions. Solutol 10 15 20 25 30 35 40 45 G' and G'' (Pa) 10 -3 10 -2 10 -1 Soluplus Temperature (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 water G' G'', 12% in PBS G' G'', 20% in water G' G'', 20% in PBS G'', 12% in water G'', 12% in PBS G'', 20% in water G'', 20% in PBS ÁNGELA VARELA GARCÍA 96 Differently, acyclovir solubility in 20% w/w Solutol micelles showed a minor increase, being 1.54 (s.d. 0.03) and 1.39 (s.d. 0.06) mg/mL in PBS and water, respectively (Figure 3.3). Figure 3.3. Apparent solubility of acyclovir in micelle dispersions of Soluplus and Solutol HS 15, prepared in water and PBS pH 7.4. Error bars represents standard deviation (n=3). Nevertheless, compared to other drugs formulated in these polymeric micelles, the total increase in solubility was relatively low, with may be related to the polarity of acyclovir (LogP= -1.56) (Al- Ghabeish et al., 2015; Hou et al., 2016). Free energy of solubilization was negative in all cases, which means that the solubilization occurred spontaneously and was thermodynamically favored by the hydrophobicity of micelle core. Previous works have reported on solubilization values of up to 15 Copolymer concentration (% w/w) 0 4 8 12 16 20 Acyclovir solubilized (mg/mL) 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 Soluplus-Water Soluplus-PBS Solutol-Water Solutol-PBS 3. Polymeric micelles for acyclovir ocular delivery: formulations and cornea and sclera permeability 97 mg/ml using O’O-lauroyl chitosan (Tan, 2016). Despite of having similar HLB values, Soluplus composition and architecture as a selfassembled copolymer seem to be more suitable to host acyclovir than the Solutol mixture of poly (ethylene oxide) esters of 12- hydroxystearic acid and free polyethylene glycol. Therefore, Solutol was discarded for subsequent studies. Parameters used to quantify the efficiency of solubilization are summarized in Table 3.2. The micelle-water partition coefficient (P) for Solutol dispersions was below 1, which means that there are more acyclovir molecules solubilized in the aqueous medium than inside the micelles. Differently, an increase in the partition coefficient values was observed for Soluplus as the copolymer concentration increased, indicating that for micelle systems prepared with Soluplus at 16% or higher the amount of acyclovir into the micelles surpasses the amount solubilized in the outer aqueous medium. ÁNGELA VARELA GARCÍA 104 Figure 3.6. Accumulated amounts of acyclovir permeated through bovine sclera during 6 h, for aqueous solution of ACV (0.3 mg/mL) and formulation of ACV in Soluplus 20% micelles (1.34 mg/mL), at 37 ºC. The results showed 10 times greater amounts of drug permeated through the sclera than through the cornea. Since the experiments were carried using similar surface area for both tissues, the differences are clearly related to the greater permeability of sclera, which has a relatively porous structure that allows drug diffusion either as free molecules or after being encapsulated in micelles (Ahmed et al., 1987; Hamalainen et al., 1997; Tai et al., 2003; Wen et al., 2010; Wen et al., 2013). Previous studies with cyclosporine encapsulated in Pluronic/TPGS mixed micelles showed that the drug accumulated in the sclera (probably because hydrophobic interactions) and only a small fraction permeated through the receptor (Grimaudo et al., 2018). Differently, in the case of acyclovir-Soluplus micelles, there was also Time (h) 0123456 Acyclovir permeated (g/cm 2 ) 0 20 40 60 80 100 120 140 160 180 ACV ACV-Soluplus 3. Polymeric micelles for acyclovir ocular delivery: formulations and cornea and sclera permeability 105 sclera accumulation but most drug could readily diffuse through it, which may favor the access to the posterior eye segment. Compared to acyclovir free in solution, encapsulation in Soluplus micelles was advantageous both in terms of (i) total amount of acyclovir accumulated in the sclera, which was 2.02 µg/cm2 (s.d. 1.61) when applied as aqueous solution, and 13.69 µg/cm2 (s.d. 4.10) when formulated in Soluplus micelles; and (ii) steady state flux (J) obtained from the slope of the amount permeated through the sclera vs. time, which was 8.02 and 26.97 µg/cm2·h-1 for acyclovir aqueous solution and micelle formulation, respectively. 3.4. Concluding remarks Soluplus micelles loaded with acyclovir showed a homogeneous nanometer particle size with slightly negative Z-potential values, which may be adequate to cross cornea and sclera. In addition, this system was shown to be suitable for formulation as eye drops that may undergo in situ gelling since the storage and loss moduli increase as temperature raise from room temperature to ocular temperature. The increase in viscosity of the formulation would prolong its permanence on the eye surface, slowing down the dilution process. The encapsulation of the antiviral drug did not greatly improve its apparent solubility, but it did notably facilitate the penetration through and accumulation in the eye tissues. In particular, the noticeable accumulation of acyclovir at the sclera level may facilitate the access of this drug to the posterior segment of the eye. ÁNGELA VARELA GARCÍA 106 3.5. References Ahmed I., Gokhale R.D., Shah M.V., Patton T.F. 1987. 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Among the proposed procedures to endow the hydrogel CLs with affinity for specific molecules, the creation of artificial receptors using the molecular imprinting technique stands out (Byrne et al., 2002; Alvarez-Lorenzo et al., 2004). This technique requires incorporating the substances of interest into the monomers mixture so that the monomers can rearrange according to their affinity. This rearrangement becomes permanent during polymerization. The removal of the template molecules generates cavities with the most appropriate size and chemical groups to host the substance of interest again (Alvarez-Lorenzo et al., 2004). Functional monomers suitable for interaction with the antiviral drugs were first screened using computational modeling; methacrylic acid (MAA) showed higher affinity for the drugs than the structural monomer 2-hydroxyethyl methacrylate (HEMA) and other functional monomers (Figure 4.1). MAA may interact with the side chain of VACV through not only hydrogen bonding with the ring (as in the case of ACV) but also electrostatic interactions with the amino group. Hydrogels were prepared with various contents in the functional monomer in the presence (imprinted) and absence (non-imprinted) of the drug. The hydrogels will be characterized in terms of swelling, transmittance, mechanical properties, biocompatibility (HET-CAM assay) and 4. Imprinted hydrogels for acyclovir and valacyclovir ocular administration 121 capability to load and release the antiviral drugs. Finally, the permeability through bovine cornea and sclera of the drug released by the hydrogel was evaluated. Figure 4.1. Structure of methacrylic acid, acyclovir and valacyclovir. 4.2. Materials and methods 4.2.1. Materials Acyclovir was purchased from Farmalabor (Italy); valacyclovir, 2,2′-azo-bis(isobutyronitrile) (AIBN), dichlorodimethylsilane, ethyleneglycol dimethacrylate (EGDMA) and methacrylic acid (MAA) were from Sigma-Aldrich (Germany); ethanol absolute and NaOH were from VWR (Belgium); 2-hydroxyethyl methacrylate (HEMA) was from Merck (Germany); acetic acid and NaCl were from Scharlau (Spain); and methanol was from Fisher (Belgium). Ultrapure water (resistivity > 18 MΩ·cm) was obtained by reverse osmosis (MilliQ®, Millipore Spain). Simulated lacrimal fluid (SLF) was prepared with the following composition: 6.78 g/L NaCl, 2.18 g/L NaHCO3, 1.38 g/L KCl and 0.084 g/L CaCl2·2H2O with pH 7.5. Carbonate buffer pH 7.2 was prepared by mixing buffer solution A Methacrylic acid Valacyclovir Acyclovir 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 buffer solution B (0.115 g/L CaCl2 and 0.155 g/L MgCl2·6H2O). 4.2.2. Computational modeling A preliminary study was carried out using computer modeling to elucidate interactions between the drugs to be studied (ACV and VACV) and functional monomers used in the synthesis of hydrogels. The tested monomers were acryl amide (AAm), 2-aminoethyl methacrylate hydrochloride (AEMA), N-(3-aminopropyl) methacrylamide hydrochloride (APMA), ethyleneglycolphenylether methacrylate (EGPEM), butoxyethyl methacrylate (BEM), hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAAc). The 3D structure of the functional monomers and ACV and VACV was taken from the PubChem database (Kim et al., 2016). The Autodock Tools version 4.2.6 software was used to calculate molecular docking. In all cases, the grid was generated with default settings around the monomer and the drug, the smallest conformation was used and the docking was performed using the Lamarckian Genetic Algorithm (Morris et al., 2009). Estimated free energy of binding (  Gbinding) and dissociation constant (Ki) values were obtained. 4.2.3. Synthesis of imprinted hydrogels Different mixtures of monomers were prepared, as shown in Table 4.1. The components were added to vials and mixed at room 4. Imprinted hydrogels for acyclovir and valacyclovir ocular administration 123 temperature and under magnetic agitation (300 rpm) until they were completely dissolved. Finally, the initiator (AIBN) was added, and the solutions were stirred for 15 minutes more. The solutions were injected, with needle and syringe, into pre-assembled moulds, consisting of two pre-treated glass plates (12x14 cm) separated by a 0.45 mm thick silicone frame. The pre-treatment of the glass consisted of applying two layers of dichlorodimethylsilane, waiting 10 minutes between each application. The plates were left to dry in a hood for 1 hour and then were thoroughly washed with ethanol and rinsed with water. Finally, they were dried in an oven at 70ºC for 1 hour, before being assembled. Polymerization was carried out for 12 hours at 50°C and then for a further 24 hours at 70°C. All hydrogel compositions were prepared in triplicate. ÁNGELA VARELA GARCÍA 124 Table 4.1. Composition of the synthesized hydrogels (NIP: non-imprinted hydrogels, MIP: imprinted hydrogels). Final EGDMA, MAA and AIBN concentrations were 8, 200 and 10 mM, respectively. Hydrogel 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. Drug removal After polymerization, each hydrogel sheet was immersed in 500 mL of boiling water for 15 minutes in order to remove unreacted monomers and facilitate cutting into discs (10 mm in diameter). Washing was then performed, except for a few discs of each type of hydrogel, which were reserved for direct drug release test, as explained below. For the washing, the discs were immersed in water, under magnetic agitation (300 rpm) and at room temperature. The media was replaced every 24 hours until no signal was measured, which was monitored spectrophotometrically in the range of 190-800 nm (UV-Vis spectrophotometer, Agilent 8534, Germany). When no 4. Imprinted hydrogels for acyclovir and valacyclovir ocular administration 125 spectrophotometric signal was detected, the hydrogels were dried in an oven at 70°C for 24 hours and stored protected from light and humidity. In parallel, the amount of ACV (MIPACV, MIP1:5, MIP1:10 and MIP1:15) and VACV (MIPVACV, MIP1:6. MIP1:12 and MIP1:32) removed in each washing step was monitored spectrophotometrically at 252 and 253 nm, respectively. 4.2.5. Direct drug release test from boiled hydrogels Discs of each type of hydrogel were individually placed in 5 mL of SLF and kept under oscillating agitation (300 rpm) and at 35 ºC. At preset times (0.5, 1, 2, 6 and 24 h), 3 mL of medium were removed and absorbance was measured by spectrophotometry at 252 nm (ACV) and 253 nm (VACV) (UV-Vis spectrophotometer, Agilent 8453, Germany), returning the samples to the release vial. The experiments were carried out in triplicate. The amounts of drug released were calculated using the previously prepared calibration curves and referred to the unit of mass of the dry disc. The calibration curves were prepared by dissolving ACV (30 µg/mL) in ethanol:water (50:50, v/v) mixture, and VACV (50 µg/mL) in water. Dilutions of 2, 3, 5, 10, 15, 20, 25 and 30 µg/mL were made for ACV, and 1.25, 2.5, 5, 10, 15, 20, 25, 30, 40 and 50 µg/mL for VACV. The calibration curve was prepared from absorbances recorded at 252 and 253 nm, respectively (UV-Vis spectrophotometer, Agilent 8453, Germany). ÁNGELA VARELA GARCÍA 126 4.2.6. Drug loading and release from conditioned hydrogels The ACV load was tested on the hydrogels NIP0, NIP200, MIPACV, MIP1:5, MIP1:10 and MIP1:15. Washed and dry discs of each type were placed in triplicate in tubes with 15 mL of drug solution. The loading solution was prepared by dissolving ACV (0.3 mg/mL) in water and kept under magnetic agitation (300 rpm), at room temperature for 72h. The loading tubes were kept under oscillating agitation (300 rpm), at RT (23-25 ºC), for 4 days. The absorbances of each sample were measured by spectrophotometry at 252 nm (UV-Vis spectrophotometer, Agilent 8453, Germany), diluting the sample (0.2:5) with ethanol:water mixture (50:50, v/v). The calculation of the total drug load on the discs was estimated by the difference between the initial and final amount of drug in solution, and was calculated using the previously prepared calibration curve, referring the loaded amounts to the unit of mass of the dry disc. The VACV load was evaluated on the hydrogels NIP0, NIP200, MIPVACV, MIP1:6, MIP1:12 and MIP1:32. Three dry discs of each type were placed in tubes with 5 mL of drug solution. The loading solution was prepared by dissolving VACV (0.3 mg/mL) in a 0.1 mM NaOH solution and was kept under magnetic agitation (300 rpm), at RT (23- 25 ºC), for 15 minutes. The loading tubes were kept under the same conditions of agitation, temperature, and time as for the ACV. The absorbances of each sample were measured spectrophotometrically, at 253 nm, for the calculation of the total load, in the same way. 4. Imprinted hydrogels for acyclovir and valacyclovir ocular administration 127 The drug network/water partition coefficient (KN/W) was calculated for each hydrogel from the total amount of drug loaded using the following equation: 𝐿𝑜𝑎𝑑𝑖𝑛𝑔 (𝑡𝑜𝑡𝑎𝑙)= ∗ ∗𝐶 [Eq. 4.1] where Vs is the volume of water absorbed by the hydrogel, Vp the volume of dry polymer, Wp the weight of the dry hydrogel, and C0 the concentration of drug in the loading solution. The loaded discs were removed from the tubes and rinsed with water. The surface water was removed with filter paper and then the discs were immediately placed in release tubes with 15 and 10 mL of SLF (for ACV and VACV, respectively), under oscillating agitation (300 rpm) and at 35 ºC. The release kinetics were evaluated at 1, 2, 4, 6, 8 and 24 h, for ACV, and at 0.5, 1, 2, 3, 4, 5, 6, 10 and 24 h, for VACV. The samples were taken following the same protocol as in section 4.1.4. 4.2.7. Degree of swelling The degree of swelling was monitored in water and SLF for hydrogels NIP0, NIP200, MIPVACV, MIP1:6, MIP1:12 and MIP1:32, in triplicate. The study was carried out at RT (23-25 ºC) and the increment in weight of the hydrogels was recorded at predetermined times (0.5, 1, 2, 4, 8 and 24 h), after being submerged in 4 mL of the corresponding medium. In each measurement, the disc was removed from the vial, superficially dried with filter paper, weighed and ÁNGELA VARELA GARCÍA 128 returned to the vial. The degree of swelling was calculated with the following equation: 𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑑𝑒𝑔𝑟𝑒𝑒 (%)= ∗100 [Eq. 4.2] where W0 and Wt represent the weight of the dried and swollen hydrogel, respectively. 4.2.8. Light transmittance The light transmission (%) of the hydrated discs in the swelling test (SLF) was measured in a spectrophotometer (Agilent Cary 60 UV-Vis) in triplicate, recording the transmittance from 200 to 800 nm. 4.2.9. Mechanical properties The mechanical properties of the NIP0, NIP200, MIPVACV, MIP1:6, MIP1:12 and MIP1:32 discs, swollen in water, were tested in triplicate at RT (23-25 ºC). Each hydrogel was cut into 16 x 9 mm strips and attached to the upper and lower clamps, with a 7 mm gap, on a TA.XT Plus Texture Analyzer (Stable Micro Systems, Ltd., UK), equipped with a 5 kgf load cell. The crosshead speed which the stress-strain plots were recorded was 0.1 mm s-1. For the calculation of Young's modulus (E), the slope of the straight line part of the tensile strength (force per cross-sectional area) and the engineering stress (change of active length divided by original length) were used, as follows (Tranoudis et al., 2004; Bhamra et al., 2017). 𝐸 =   ∆   [Eq. 4.3] 4. Imprinted hydrogels for acyclovir and valacyclovir ocular administration 129 4.2.10. HET-CAM test The chorioallantoic membrane hen egg test (HET-CAM) was performed by incubating fertilized hen eggs (50-60 g) at 37°C and 60% RH for 9 days. On the ninth day of incubation a circular cut was made on the top of the egg of approximately 1 cm diameter with a rotary saw (Dremel 300, Breda, The Netherlands). The shell was removed, and the inner membrane was moistened with 0.9% NaCl for 30 min (time during which the egg remained inside the climatic chamber). The membrane was then removed to expose the chorioallantoic membrane (CAM) (Alvarez-Rivera et al., 2019). The test was performed by placing in each CAM a hydrogel of each type in triplicate, previously hydrated for 24 h in loading solution. An aqueous solution of NaOH 0.1N and NaCl 0.9% (300 µL) in triplicate was used as negative and positive controls, respectively. The blood vessels were observed under white light for 5 min, to detect possible bleeding, vascular lysis or coagulation. 4.2.11. Bovine corneal and scleral permeability test The fresh bovine eyes were collected from the local slaughterhouse and transported according to the BCOP test protocol (OECD, 2009; Alvarez-Rivera et al., 2019). During transport, the eyes were kept immersed in PBS with added antibiotics (penicillin 100 IU/ml and streptomycin 100 μg/ml), in an ice bath. The corneas and scleras were isolated, using a scalpel. The tissues were washed with 0.9% NaCl and mounted in vertical diffusion cells (Franz cells). To ÁNGELA VARELA GARCÍA 232 7. Patent 233 ÁNGELA VARELA GARCÍA 234 7. Patent 235 ÁNGELA VARELA GARCÍA 236 Abbreviations 8. Abbreviations 239 8. Abbreviations  AAPH 2,2′-azobis(2-amidino-propane) dihydrochloride  ACV Acyclovir  AFR Africa  AIBN 2,2′-azo-bis(isobutyronitrile)  AMD Age-related macular degeneration  AMR America  BAB Hemato-aqueous barrier  BCOP Bovine cornea opacity test  BRB Hemato-retinal barrier  CAT Catalase  CD Cyclodextrins ÁNGELA VARELA GARCÍA 240  CLs Contact lenses  CMC Critical micellar concentration  CMV Citomegalovirus  CO Corneal opacities  DES Dry eye syndrome  DME Macular edema  DR Diabetic retinopathy  EDTA Ethylenediaminetetraacetic acid  EGPEM Ethylene glycol-phenyl ether methacrylate  EMR Eastern Mediterranean  EUR Europe  GMA Glycidyl methacrylate  GPx Glutathione peroxidase  HCEC Human corneal epithelial cells  HEMA Hydroxyethyl methacrylate 8. Abbreviations 241  HET-CAM Hen’s egg test on chorio-allantoic membrane  HLB Hydrophilic-lipophilic balance  HSV Herpes simplex virus  IOP Intraocular pressure  MAA Methacrylic acid  MW Molecular weight  ORAC Oxygen radical antioxidant capacity  PBS Phosphate buffered saline  PDI Polydispersion index  PLGA Poly lactic-co-glycolic acid  PVR Proliferative vitreoretinopathy  RE Refractive errors  RNS Reactive nitrogen species  ROS Reactive oxygen species  RT Room temperature