Hydrogels as platforms for vaccine delivery and mucosal restoring

DOCTORAL THESIS HYDROGELS AS PLATFORMS FOR VACCINE DELIVERY AND MUCOSAL RESTORING Lorena García del Río INTERNATIONAL DOCTORAL SCHOOL DOCTORAL PROGRAM IN DRUG RESEARCH AND DEVELOPMENT SANTIAGO DE COMPOSTELA 2021 TESE DE DOUTORAMENTO HIDROXEIS COMO PLATAFORMAS PARA A ADMINISTRACIÓN DE VACINAS E REPARACIÓN DA MUCOSA Lorena García del Río ESCOLA INTERNACIONAL DE DOUTORAMENTO PROGRAMA DE DOUTORAMENTO EN INVESTIGACIÓN E DESENVOLVEMENTO DE MEDICAMENTOS SANTIAGO DE COMPOSTELA 2021 PhD CANDIDATE STATEMENT Hydrogels as platforms for vaccine delivery and mucosal restoring Miss Lorena García del Río 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 applicable, explicit mention is given to the collaborations the work may have had. 3) The present document is the final version submitted for its defence and coincide with the document sent in electronic format. 4) I confirm that this 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 ...............................................2021 Sgd. Lorena García del Río DECLARACIÓN DO AUTOR DA TESE Hidroxeis como plataformas para a administración de vacinas e reparación da mucosa Dna Lorena García del Río Presento a miña tese, seguindo o procedemento axeitado ao Regulamento, e declaro que: 1) A tese abarca os resultados da elaboración do meu traballo. 2) De ser o caso, na tese faise referencia ás colaboracións que tivo este traballo. 3) Confirmo que a tese non incorre en ningún tipo de plaxio doutros autores nin de traballos presentados por min para a obtención doutros títulos. 4) A tese e a versión definitiva presentada para a súa defensa e coincide a versión impresa coa presentada en formato electrónico. E comprométome a presentar o Compromiso Documental de Supervisión no caso de que o orixinal non estea na Escola. En Santiago de Compostela, a .............................................2021 Sgd. Lorena García del Río AUTHORIZATION OF THE THESIS SUPERVISOR Hydrogels as platforms for vaccine delivery and mucosal restoring Prof. Mariana Landín, as director and tutor REPORT: That the present thesis, corresponds to the work carried out by Miss Lorena García del Río, under my supervision, and that I authorize its presentation considering that it gathers the necessary requirements of the USC Doctoral Studies Regulation, and that as supervisor of this thesis, it does not incur in the abstention causes established by the Law 40/2015. According to the Regulation of Doctorate Studies, I also declare that the present doctoral thesis is suitable to be defended on the basis of the Monographic modality with reproduction of publications, in which the participation of the doctoral student was decisive for its elaboration and that the publications correspond to the research plan. At Santiago de Compostela, on ......................................2021 Sgd. Mariana Landín Pérez Chegado este momento e botando unha ollada atrás no tempo, podo dicir con total seguridade que a miña primeira incursión no mundo da investigación veu a raíz da miña curiosidade por querer saber sempre o porqué das cousas e, a un golpe de sorte. Estar no momento e lugar adecuado fixo posible que hoxe estea escribindo estas palabras de agradecemento. Por iso, en primeiro lugar, creo que debo de comezar pola persoa que me abriu as portas do mundo da investigación de par en par, Mariana Landín. Sen a oportunidade e a confianza que me brindou fai xa oito anos, a realización deste traballo non tería sido posible. Grazas a ela, o que a priori ía ser unha pequena aventura científica, rematou sendo toda unha carreira de fondo na que sempre intentou apoiarme como unha directora próxima, liberal, comprensiva e sobre todo, moi paciente. Baixo as súas directrices puiden experimentar unha gran evolución, tanto a nivel persoal coma académica. En segundo lugar, non podía faltar Patricia Díaz Rodríguez, unha persoa extraordinaria en tódolos aspectos. Traballar con ela é un auténtico pracer, sempre cunha enerxía desbordante, contaxiando a súa paixón pola ciencia e disposta a botar unha man sen esperar nada a cambio. Durante todo este tempo sempre estivo aí, incluso desde a distancia, para escoitarme, aconsellarme e guiarme nos momentos máis incertos. Moitas grazas por todo Patri! A tódolos meus compañeiros de laboratorio por facer máis amena esta experiencia, así como a Fani, Cris e “os de Fran”. Aínda que coincidimos case o final desta etapa, gustaríame agradecerlle a Rebeca as súas palabras de ánimo, simpatía e amizade desinteresada. Especial mención merecen Helena, Leti, Jorge, Víctor, Mirian e Jesús que acabaron converténdose en bos amigos. Abofé que esta andanza non sería a mesma sen o seu apoio, bo humor e esas grandes vivencias compartidas dentro e fóra do laboratorio; abofé que non. Ós profesores Carmen Álvarez Lorenzo, Ángel Concheiro Nine e Francisco Otero Espinar pola súa boa disposición e, por sempre sacar un oco para intentar axudarme con certas dúbidas que foron xurdindo ó longo da tese, así como a Profesora María del Carmen Remuñán por deixarme o seu laboratorio a miña total disposición. Ocupan un lugar destacado os doutores Dennis Christensen e Gabriel Kristian Pedersen do Statens Serum Institut, que apostaron por este proxecto e recibíronme cos brazos abertos sen coñecerme de nada. Grazas a eles puiden explorar outros métodos de traballo e mergullarme no mundo da inmunoloxía dunha maneira amena, atractiva e “relativamente” fácil. A Rune e Julia pola súa amabilidade. A Gregers, Frank, Joshua, Jes e Álvaro, por esas charlas tan interesantes e divertidas. Ós meus compañeiros do SSI, Nida, Nina, Julie, Dennis, Katharina, Elisa e Safiya, por integrarme no grupo desde o primeiro momento e permitirme coñecela sociedade danesa dunha maneira afable e próxima. Máis aló do laboratorio, tampouco poden faltar as persoas que contribuíron a que a miña estadía en Copenhague fose algo incrible e inesquecible. Grazas Kike, Vanessa, Federica, Cris, Andrea Arias, Andrea Castaño, Guido, Paula e Flor, por tódalas experiencias vividas e por ser a miña familia no frío norte. Tamén me gustaría agradecer a esas persoas que estiveron ó meu lado todos estes anos, ben desde os inicios ou que se foron incorporando ó longo desta andanza, mostrándome a súa amizade e apoio moral nos momentos máis escuros. Grazas a Sheila, Ana, Laura, Vero, Cris, Jessi e Gabriele por estar sempre dispostos a buscar unha boa escusa para ir de troula e así axudarme a desconectar da mellor maneira posible. Non podo esquecerme de Gabriela, a cal é a miña mellor amiga e unha das mellores persoas coas que puiden coincidir na carreira. Grazas por esas cañas; esas esmorgas con final imprevisible e a veces rozando o surrealista; e por compartir comigo unha das miñas grandes afeccións, viaxar e descubrir mundos novos. Por último, pero non menos importante, teño que darlle as GRAZAS a miña familia. Sobre todo a miña nai, Lourdes. Por ela puiden subirme a este tren, xa que sen o seu esforzo, cariño, comprensión, paciencia e ganas de que me converta na mellor versión de min mesma, nada disto sería posible. A meu irmán, Jorge, por ese humor irónico, a veces un tanto negro, que en moitas ocasións conseguiu sacarme un gran sorriso. E finalmente, a miña tía Concha, por estar sempre presente e ser coma unha segunda nai. Grazas a todos, porque sen a vosa contribución esta experiencia non sería igual de enriquecedora! TABLE OF CONTENT Table of content CONTENT List of abbreviations ..................................................................................... I Resumo/Abstract ........................................................................................ IX Resumo in extenso .................................................................................. XVII Expanded abstract ........................................................................... XXXVII 1. Introduction .............................................................................................. 1 1.1. Mucosal surfaces ..................................................................................... 3 1.1.1. Anatomy and function (small overview) .......................................... 3 1.1.2. Mucus and mucins ............................................................................ 5 1.2. Inmunity from mouth to rectum .............................................................. 6 1.2.1. Oral cavity immune system .............................................................. 6 1.2.2. Gut-associated lymphoid tissues or GALT ...................................... 7 1.3. Mucosal vaccination: pros and cons ...................................................... 10 1.3.1. Adjuvants ....................................................................................... 11 1.3.2. Mucosal tissues as vaccination sites ............................................... 12 1.3.2.1. Ocular vaccination................................................................... 13 1.3.2.2. Nasal and pulmonary vaccination ........................................... 14 1.3.2.3. Sublingual and buccal vaccination .......................................... 15 1.3.2.4. Oral vaccination ...................................................................... 16 1.3.2.5. Vaginal and rectal vaccination ................................................ 17 1.4. Gastrointestinal inflammatory mucosal pathologies ............................. 17 1.5. Hydrogels: types and properties ............................................................ 20 1.5.1. Stimuli-sensitive hydrogels ............................................................ 24 1.5.1.1. Physically responsive hydrogels ............................................. 24 1.5.1.2. Chemically and biochemically responsive hydrogels ............. 27 1.5.2. Hydrogels as drug delivery platforms on mucosal surfaces ........... 30 LORENA GARCÍA DEL RÍO 1.5.2.1. Formulation of hydrogels with desirable characteristics ......... 30 1.6. Artificial intelligence techniques applied to pharmaceutical systems design ............................................................................................................ 32 References .................................................................................................... 36 2. Objectives ................................................................................................ 61 References .................................................................................................... 65 3. Chapter I: “Design of novel orotransumucosal vaccine delivery platforms using artificial intelligence” ..................................................... 67 3.1. Introduction ........................................................................................... 71 3.2. Materials and methods ........................................................................... 73 3.2.1. Materials ......................................................................................... 73 3.2.2. Proliferation assays in human macrophages ................................... 74 3.2.3. Experimental design ....................................................................... 74 3.2.4. Hydrogels preparation and characterization ................................... 75 3.2.4.1. Rheological analysis ................................................................ 76 3.2.4.2. Texturometric assessments ...................................................... 76 3.2.5. Antigen-like microspheres loading and in vitro release studies ..... 77 3.2.6. Artificial Neural Networks modelization ........................................ 77 3.2.7. Statistical analysis .......................................................................... 78 3.3. Results ................................................................................................... 79 3.3.1. Immunostimulatory capacity of selected polymers ........................ 79 3.3.2. Hydrogels performance and AIT modelling ................................... 80 3.3.2.1. Tgel is mainly determined by PF127 and Gantrez® concentration .............................................................................................................. 81 3.3.2.2. Mucoadhesion, adhesion work and gel strength are dependent on proportions of PF127 and Gantrez® ................................................. 82 3.3.2.3. Cohesion is modulated by PF127 ............................................ 83 Table of content 3.3.2.4. GFM released at 5 min was fundamentally modulated by PF127 while total release time is also dependent on type of Gantrez®............ 84 3.4. Discussion ............................................................................................. 86 3.5. Conclusions ........................................................................................... 89 References .................................................................................................... 90 4. Chapter II: “Sublingual boosting with a novel mucoadhesive thermogelling hydrogel following parenteral CAF01 priming as strategy against Chlamydia trachomatis”............................................................... 97 4.1. Introduction ......................................................................................... 101 4.2. Materials and Methods ........................................................................ 103 4.2.1. Materials ....................................................................................... 103 4.2.2. Hydrogel development and preparation ....................................... 104 4.2.3. Hydrogel characterization ............................................................ 105 4.2.3.1. Rheological properties ........................................................... 105 4.2.3.2. Texturometric properties ....................................................... 106 4.2.3.3. Protein release ....................................................................... 106 4.2.3.4. Stability properties ................................................................ 107 4.2.4. CAF01 liposomes preparation ...................................................... 107 4.2.5. Antigen and formulations loading ................................................ 107 4.2.6. In vivo assays ............................................................................... 108 4.2.6.1. Ethics statement..................................................................... 108 4.2.6.2. Mouse immunization ............................................................. 108 4.2.6.3. Antibody titers in blood and vaginal samples ....................... 109 4.2.6.4. IFN-γ and IL-17A responses in spleen and cervical lymph nodes .................................................................................................. 110 4.2.7. Statistical analysis ........................................................................ 111 4.3. Results ................................................................................................. 111 LORENA GARCÍA DEL RÍO 4.3.1. Hydrogel preparation, characterization and stability .................... 111 4.3.2. Prime immunization using CAF01 followed by OGEL mucosal boosting improves mucosal antibody responses ..................................... 112 4.3.3. Prime immunization using CAF01 followed by OGEL sublingual boosting elicits high-magnitude IFN-γ responses in cervical lymph nodes ................................................................................................................ 114 4.4. Discussion............................................................................................ 115 References .................................................................................................. 119 5. Chapter III: “New tools to design smart thermosensitive hydrogels for protein rectal delivery in IBD” ................................................................ 127 5.1. Introduction ......................................................................................... 131 5.2. Materials and methods ......................................................................... 132 5.2.1. Experimental designs .................................................................... 132 5.2.2. Hydrogel preparation .................................................................... 134 5.2.3. Texturometric analysis ................................................................. 134 5.2.4. Rheological assessment ................................................................ 135 5.2.5. Protein loading and in vitro release studies .................................. 135 5.2.6. Data modelization using Artificial Neural Networks ................... 136 5.3. Results ................................................................................................. 137 5.3.1. Knowledge space analysis ............................................................ 137 5.3.2. Design space analysis ................................................................... 140 5.3.2.1. PF127 determines syringeability ........................................... 142 5.3.2.2. PF127 and MK4M rule bioadhesiveness ............................... 142 5.3.2.3. PF127 and MK4M determine viscosity ................................. 142 5.3.2.4. PF68 rules mainly the Tgel ..................................................... 144 5.3.2.5. In vitro protein release studies ............................................... 144 5.4. Discussion............................................................................................ 145 Table of content 5.5. Conclusions ......................................................................................... 149 References .................................................................................................. 150 6. Chapter IV: “Tailored hydrogels as delivery platforms for conditioned medium from mesenchymal stem cells in a mouse model of acute colitis” .............................................................................................. 157 6.1. Introduction ......................................................................................... 161 6.2. Materials and methods ........................................................................ 162 6.2.1. Materials ....................................................................................... 162 6.2.2. Hydrogel development and preparation ....................................... 163 6.2.3. Hydrogel Characterization ........................................................... 164 6.2.3.1. Texturometric analysis .......................................................... 164 6.2.3.2. Rheological analysis .............................................................. 164 6.2.3.3. Stability assessments ............................................................. 164 6.2.4. Cell Cultures and hUCESC-CM preparation ............................... 165 6.2.5. Hydrogel loading with hUCESC-CM .......................................... 165 6.2.6. In vivo assay ................................................................................. 165 6.2.6.1. Ethics statement..................................................................... 165 6.2.6.2. Mice ....................................................................................... 165 6.2.6.3. Colitis induction and experimental groups ............................ 166 6.2.6.4. Clinical symptoms evaluation and colon macroscopic examination ........................................................................................ 167 6.2.6.5. Histological evaluation .......................................................... 167 6.2.6.6. RNA Extraction and Quantitative Real Time PCR ............... 167 6.2.7. Statistical analysis ........................................................................ 168 6.3. Results ................................................................................................. 168 6.3.1. Hydrogel preparation and characterization .................................. 168 6.3.2. Inducement of DSS-associated colitis in C57BL/6 mice ............. 170 LORENA GARCÍA DEL RÍO VI IL-2: Interleukin 2 IL-6: Interleukin 6 kDa: Kilo Dalton LALT: Larynx-associated lymphoid tissue LCs: Langerhans cells LCST: Low critical solution temperature LDALT: Lacrimal duct-associated lymphoid tissue LOG: Blinded expert pathologist LOOCV: Leave One Out Cross Validation M cells: Microfold cells MALT: Mucosal-associated lymphoid tissues mJ: Millijoules MK4M: Methocel® K4M MLD: Minimum Description Length MSCs: Mesenchymal stem cells MZ: Mesalazine group N: Newton NALT: Nasopharynx associated lymphoid tissue NLRs: Nucleotide binding oligomerization domain- (NOD-) like receptors List of abbreviations VII NT: No-treated group OGEL: Sublingual optimized hydrogel PBS: Phosphate-buffered saline pcDNA3-CRT/E7 DNA vaccine: DNA vaccine encoding the early protein E7 from human papillomavirus type 16 linked to calreticulin PEO: Polyethylene oxide PF127: Pluronic® F127 PF68: Pluronic® F68 PLGA: Poly lactic-coglycolic acid PMA: 2-mercaptoethanol and phorbol 12-myristate 13-acetate PPO: Polypropylene oxide Q: Swelling ratio QbD: Quality by design ROS: Reactive oxygen species RLRs: Retinoic acid-inducible gene-I- (RIG-I-) like receptors rMOMP: recombinant major outer membrane protein RPMI: Roswell Park Memorial Institute medium S97: Gantrez® S97 SCoF: Simulated colonic fluid SED: Subepithelial dome LORENA GARCÍA DEL RÍO VIII SEM: Standard error of the mean sIgA: Secretory IgA antibodies SRM: Structural risk minimisation SSF: Simulated salivary fluid TDB: Glycolipid trehalose 6,6´-dihibenate Tgel: Gelation temperature TLRs: Toll-like receptors TNF-α: Tumour necrosis factor alpha UC: Ulcerative colitis UCST: Upper critical solution temperature WST-1: Water Soluble Tetrazolium Salt-1 ξ: Mesh size ρx: Crosslinking density RESUMO/ABSTRACT Resumo/Abstract XI RESUMO A administración de moléculas activas na mucosa, como as proteínas, é unha aproximación moi atractiva e de indubidables vantaxes para inducir unha resposta local e/ou sistémica de carácter terapéutico e/ou inmunoestimulante. Non obstante, aspectos críticos como a drenaxe rápida e a degradación mediada por encimas ou o pH, limitan o tempo de permanencia das moléculas terapéuticas sobre a superficie das mucosas e tamén condicionan o paso de substancias a través destes tecidos, e polo tanto, a súa utilidade. Os hidroxeis son redes poliméricas en 3D cuxas características poden modularse para lograr formulacións altamente mucoadhesivas, capaces de interactuar coas mucosas e incrementalo tempo de permanencia das proteínas no lugar de acción, conseguindo a súa absorción e o efecto terapéutico desexado. Ademais, cando se combinan características como biocompatibilidade, mucoadhesión, termosensibilidade e a elevada capacidade de carga de fármaco dos hidroxeis, estes sistemas convértense en excelentes candidatos para superar as limitacións da administración transmucosal. Con todo, o deseño deste tipo de formas farmacéuticas implica a preparación de sistemas multicompoñente, o que supón unha tarefa desafiante no ámbito da tecnoloxía farmacéutica. Nos últimos anos as ferramentas de intelixencia artificial (AI) como as redes neuronais, a lóxica difusa ou os algoritmos xenéticos (GA) mostraron a súa utilidade para estudalos efectos das variables de composición e operación sobre as características de diferentes formas farmacéuticas. Por iso, o obxectivo deste traballo experimental é abordala utilidade das ferramentas de AI no desenvolvemento de hidroxeis multicompoñente con prestacións avanzadas. Así mesmo, explórase o potencial de combinacións ternarias de polímeros seleccionados para xerar hidroxeis termosensibles e altamente bioadhesivos para a carga e cesión de proteínas con dúas aplicacións; a vacinación sublingual e o tratamento local de úlceras colorectais na enfermidade inflamatoria intestinal (EII). Con este fin, en primeiro lugar, levouse a cabo unha selección inicial de compoñentes e, estableceuse en cada caso un deseño experimental reducido utilizando o método de densidade balanceado. LORENA GARCÍA DEL RÍO XII Iso permitiu estudar, cun número moi limitado de formulacións, tanto o espazo de coñecemento coma o de deseño dos hidroxeis ternarios. Caracterizáronse as propiedades texturometricas, reolóxicas e os perfís de cesión dos hidroxeis e, analizouse o efecto de cada compoñente nas súas características mediante o emprego combinado de redes neuronais artificiais (ANN) e técnicas de lóxica difusa. A continuación, empregáronse ANN e GA de forma combinada para modelar e seleccionar, en cada caso, unha formulación optimizada que xelifique a temperatura fisiolóxica e que presente as mellores características para a súa aplicación e mantemento sobre a mucosa correspondente, resistencia ó lavado ou á dilución e elevada mucoadhesión. Por último, realizáronse estudos en ratos. Por unha banda, no caso do hidroxel optimizado para aplicación sublingual, avaliouse a súa utilidade como portador da vacina CTH522 contra Chlamydia trachomatis. Os resultados do estudo mostraron que a estratexia máis prometedora para obter inmunidade local no tracto xenital foi a administración combinada da vacina CTH522 por vía parenteral e sublingual, utilizando respectivamente CAF01 (un sistema liposomal) e o hidroxel optimizado como portadores. Os estudos in vivo tamén demostraron que esta estratexia foi máis eficaz inducindo respostas inmunitarias celulares (Th1/Th17) e incrementou de maneira significativa o IFN-γ producido polos ganglios linfáticos cervicais, comparada coa vacinación parenteral soa. Por outra banda, avaliouse a capacidade rexenerativa e antiinflamatoria do hidroxel optimizado para administración rectal previamente cargado con medio condicionado de células nai uterinas humanas (H-hUCESC-CM) nun modelo experimental de colite aguda en rato. Os datos experimentais indican que o tratamento co hidroxel cargado diminuiu significativamente a perda de peso corporal e o acurtamento do colon, a dexeneración da mucosa colónica e os niveis de ARNm de TNF-α, IFN-γ e IL-6. Os nosos datos indican que o HhUCESC-CM alivia eficazmente a colite inducida por DSS en ratos, o que suxire que o H-hUCESC-CM podería representar unha terapia sen células moi atractiva para o tratamento local da EII. Resumo/Abstract XIII Como conclusión, cabe destacar que o emprego de ferramentas de IA facilita enormemente o desenvolvemento de sistemas complexos e multicompoñente, dado que o seu uso permite reducir considerablemente o número de experimentos, o que aforra custos e tempo. Ademais o seu uso permite adquirir un coñecemento profundo dos sistemas farmacéuticos e establecelo espazo de deseño axeitado para cada formulación. Resumo/Abstract XV ABSTRACT Mucosal administration of active molecules, such as proteins, is an attractive and advantageous approach to induce local or systemic therapeutic and/or immunostimulatory responses. Critical aspects, such as rapid protein drainage or enzymeor pH-mediated degradation, limit the residence time of therapeutic molecules on mucosal surfaces and condition the passage of substances through the mucosa and, therefore, their effectiveness. Hydrogels are 3D polymeric networks with characteristics that can be modulated to achieve highly mucoadhesive formulations, capable of interacting with mucous membranes and increasing the residence time of drugs at the target site, enhancing their absorption and the desired therapeutic effect. In this sense, thermosensitive and mucoadhesive hydrogels are of particular interest to improve the efficacy or bioavailability of proteins when administered on mucous membranes. In addition, these hydrogels are excellent candidates to overcome the limitations of transmucosal delivery due to their biocompatibility, mucoadhesion, thermosensitivity and drug loading capacity. However, the design of such dosage forms involves the preparation of multicomponent systems, which is a challenging task. In recent years, artificial intelligence (AI) tools such as neural networks, fuzzy logic or genetic algorithms (GA) have proven to be very useful in studying the effects of composition and operational variables on the characteristics of different pharmaceutical systems. Therefore, this thesis work addresses the usefulness of AI tools in the development of multicomponent hydrogels with advanced performance. Thus, we explore the potential of ternary combinations of selected polymers to generate thermosensitive and highly bioadhesive hydrogels suitable for protein loading and delivery with two applications; sublingual vaccination and local treatment of colorectal ulcers in inflammatory bowel disease (IBD). For hydrogels design, after the initial selection of components, a reduced experimental design was established using a balanced density method. This made possible to delimit both the knowledge space and the design space of ternary systems using a limited number of formulations. Texturometric and rheological properties and release LORENA GARCÍA DEL RÍO XXII previamente descritos [26-30]. Estes ensaios permitiron determinala xiringabilidade, a resistencia do xel ou dureza, a adhesión, a mucoadhesión e a cohesión dos hidroxeis a 37 °C. Ademais, utilizouse un reómetro de esforzo controlado AR1000N (TA Instruments, Reino Unido) para determinala temperatura de xelificación dos sistemas en función dos rexistros dos módulos de almacenamento (G′) e perda (G″) a diferentes temperaturas. Adicionalmente, no caso dos clisteres mediuse a viscosidade complexa de cada hidroxel a 20 ºC. Para a avaliación in vitro do perfil de cesión de fármacos a partir dos hidroxeis, utilizáronse como “fármacos” modelo microesferas fluorescentes de cor verde (GFM) similares a antíxenos no caso dos hidroxeis destinados á vacinación da mucosa ou, albumina sérica bovina no caso dos clisteres. Os hidroxeis cargados colocáronse en células de cesión cun tamaño de poro de 40 μm e determinouse a porcentaxe de microesferas ou de proteína cedida a diferentes tempos usando métodos adecuados. 2.3 MODELAXE DOS HIDROXEIS UTILIZANDO TÉCNICAS DE INTELIXENCIA ARTIFICIAL Trala caracterización dos hidroxeis, utilizouse o software FormRules® v4.03 (Intelligensys Ltd., Reino Unido) que combina redes neuronais artificiais con técnicas de lóxica difusa para modelalos resultados obtidos en función da súa composición e, así analizar o efecto que cada compoñente ten nas propiedades dos hidroxeis mediante as regras “SE-ENTÓN” que xera. Adicionalmente, empregouse o software INForm® v.5.01 (Intelligensys, Ltd. UK) que combina as redes neuronais artificiais con algoritmos xenéticos para modelar e seleccionar, en cada caso, unha formulación optimizada que xelifique a temperatura fisiolóxica e que presente as mellores características para a súa aplicación e mantemento sobre a mucosa correspondente, resistencia ó lavado ou a dilución e elevada mucoadhesión. A calidade dos modelos avaliouse en función dos coeficientes de determinación e o ANOVA que compara os valores preditos polo modelo cos valores experimentais. Ademais, os modelos obtidos validáronse mediante a produción e caracterización das formulacións optimizadas. Resumo in extenso XXIII 2.4 AVALIACIÓN DA ACTIVIDADE TERAPÉUTICA DOS SISTEMAS DESENVOLVIDOS 2.4.1 Ensaio de inmunización en ratos Realizouse un estudo en ratos femia CBF61 de 9 semanas para avaliar a utilidade do xel optimizado como portador sublingual da vacina CTH522 contra Chlamydia trachomatis, seguindo diferentes estratexias de vacinación. Para iso, os animais dividíronse en catro grupos con 3-5 ratos cada un. O esquema de inmunización móstrase na Figura 1. Do Grupo 1 ó 3 recibiron unha dose inicial do antíxeno CTH522 formulado en liposomas CAF01 [31] por vía subcutánea na base do rabo (Grupos 1, 2 e 3) e o Grupo 4 foi inmunizado co hidroxel optimizado (OXEL) cargado co mesmo antíxeno por vía sublingual. A segunda e terceira dose administráronse ós 15 e 35 días tal e como se describe a continuación. O Grupo 2 recibiu unha segunda e terceira dose do antíxeno CTH522 por vía sublingual. Os Grupos 3 e 4 recibiron unha segunda e terceira dose do hidroxel cargado coa vacina CTH522. O Grupo 1 non recibiu ningunha dose adicional. Figura 1. Esquema de inmunización e grupo de ratos testados. Os animais foron cebados por vía subcutánea (s.c) ou sublingual (s.l) usando CAF01 e OXEL como sistemas de cesión da vacina. O día 57, os animais foron sacrificados. Ó final do estudo determináronse os anticorpos específicos IgG e IgA totais fronte a CTH522 en soro e lavado vaxinal. Adicionalmente, determináronse no bazo e nos ganglios linfáticos cervicais as concentracións de IFN-γ e IL-17A mediante ELISA. As diferenzas entre os grupos experimentais LORENA GARCÍA DEL RÍO XXIV establecéronse mediante os resultados dun ANOVA dunha vía e o test de Tukey para comparacións múltiples (p<0.05). Os experimentos leváronse a cabo no Statens Serum Institut (SSI) de Dinamarca, de acordo coas regulacións establecidas polo Ministerio de Xustiza de Dinamarca e os comités de protección animal segundo o Permiso de Inspección de Experimentos con Animais Danés 2017-15-0201-01363 e de conformidade coa Directiva da Comunidade Europea 2010/63. 2.4.1 Ensaio nun modelo de rato con colite aguda Realizouse un estudo nun modelo de colite aguda en ratos macho C57BL/6 para avaliar a utilidade do clister optimizado e cargado con medio condicionado de células nai cervicais humanas, para o tratamento local de úlceras colorectais en EII. Para comprobar a utilidade dos hidroxeis cargados con medio condicionado na EII, utilizouse o protocolo descrito por Wirtz et al. (Figura 2) [32]. Induciuse unha colite aguda en ratos mediante a administración de dextrano sulfato de sodio (DSS) ó 3.5% na auga de bebida durante 9 días. Entre os días 10 e 15, administráronse dúas veces ao día 75 μl de cada tratamento por vía rectal utilizando unha agulla de punta redondeada de calibre 26. Os animais dividíronse en 5 grupos como segue: 1) Grupo de control san; 2) Grupo non tratado: ratos que non recibiron ningún tipo de tratamento tras a administración do DSS; 3) Grupo control H-DMEM-F12: ratos tratados soamente co hidroxel tras a indución da colite; 4) Grupo HhUCESC-CM: ratos tratados co hidroxel cargado con medio condicionado tras a indución da colite; e 5) Grupo mesalazina ( MZ): ratos tratados con escuma de mesalazina tras a indución da colite (tratamento habitual da EII). Ós animais practicóuselles a eutanasia no día 16 de ensaio. Resumo in extenso XXV Figura 2. Diagrama do estudo. Indución da colite nos animais usando DSS e parámetros avaliados. Avaliáronse os síntomas clínicos (supervivencia, cambios no peso corporal, consistencia das feces e severidade do sangrado) e, tras o sacrificio dos animais levouse a cabo un exame macroscópico do colon (lonxitude). Adicionalmente, tamén se levou a cabo unha avaliación histolóxica (grao de inflamación e infiltración celular, ulceracións microscópicas, arquitectura das criptas, atrofia e displasia epitelial) e a extracción de ARNm do colon para a realización de PCRs cuantitativas en tempo real para determinala expresión de TNFα, IL-6 e IFN-. Este estudo realizouse segundo o protocolo aprobado polo Comité de Ética de Experimentos con Animais da Universidade de Santiago de Compostela. 3. RESULTADOS E DISCUSIÓN 3.1 HIDROXEIS TERNARIOS, TERMOSENSIBLES E MUCOADHESIVOS COMO SISTEMAS PORTADORES DE PROTEÍNAS PARA VACINACIÓN SUBLINGUAL En primeiro lugar, proponse a combinación do PF127, HS1200 e Gantrez® (AN119 ou S97) como materias primas para producir un hidroxel de aplicación sublingual, útil para a inmunización da mucosa. Os estudos realizados para lograr este obxectivo preséntanse no Capítulo I e aparecen resumidos na Figura 3. LORENA GARCÍA DEL RÍO XXVI Figura 3. Diagrama xeral dos ensaios descritos no Capítulo I. Os estudos realizados en macrófagos indicaron un potencial efecto inmunoestimulante do HS1200, promovendo a proliferación das devanditas células presentadoras de antíxeno. Por outra banda, ningún polímero presentou toxicidade algunha. As ferramentas de AI, en particular a combinación das redes neuronais artificiais coa lóxica difusa (neurofuzzy logic) permitiron establecer o papel de cada polímero no comportamento reolóxico e texturométrico do hidroxel, así como a súa capacidade para retelas microesferas (GFM) empregadas como antíxeno modelo. Así, a interacción entre o PF127 e o Gantrez® condiciona tanto a temperatura de xelificación como os traballos de mucoadhesión e adhesión, ou a resistencia do xel. Observáronse diferenzas entre as dúas variedades de Gantrez® avaliadas, sendo a variedade S97 a que permite unha cesión das micropartículas máis prolongada. A incorporación do HS1200 na formulación deu lugar a unha redución tanto na resistencia do xel como na súa capacidade de adhesión. Utilizouse a combinación de redes neuronais artificiais con algoritmos xenéticos para modelala composición dos hidroxeis e optimizalo proceso, é dicir, seleccionar a composición ternaria que proporciona sistemas de máxima mucoadhesión, cohesión e resistencia e, que ademais xelifiquen a temperatura corporal. Os estudos realizados para lograr este obxectivo preséntanse no capítulo II e aparecen resumidos na Figura 4. Resumo in extenso XXVII Figura 4. Diagrama xeral cos ensaios descritos no Capítulo II. Elaborouse e caracterizouse o hidroxel coa composición óptima elixida polo modelo (21.1% de PF127, 5% de HS1200 e 0.08% de Gantrez® AN119). Adicionalmente, almacenouse en condicións controladas para avaliar a súa estabilidade. Tanto as características reolóxicas e texturométricas dos hidroxeis elaborados coma as preditas polo modelo concordaron completamente, o que permitiu a súa validación. Ademais, a formulación resultou ser estable. A potencial utilidade do hidroxel optimizado como portador da vacina CTH522 fronte a Chlamydia trachomatis por vía sublingual avaliouse nun modelo de rato. Os resultados do estudo mostraron que a estratexia máis prometedora para obter inmunidade local no tracto xenital foi a administración combinada da vacina CTH522 por vía parenteral e sublingual, utilizando CAF01 (sistema liposomal) e o hidroxel optimizado como portadores, respectivamente. As propiedades termosensibles do hidroxel facilitan a súa aplicación debaixo da lingua e aumentan o seu contacto co tecido sublingual; polo que a súa xelificación a temperatura corporal xunto coa rápida cesión da proteína debe evitar a súa perda ó tragar e promover a súa absorción. Os estudos in vivo tamén demostraron que a inmunización combinada parenteral-sublingual foi máis eficaz inducindo IgA no tracto xenital e respostas inmunitarias celulares (Th1/Th17) que a vacinación da mucosa ou parenteral soa. Ademais, esta estratexia LORENA GARCÍA DEL RÍO XXVIII provocou un incremento significativo do IFN-γ nos ganglios linfáticos cervicais comparada coa vacinación parenteral soa. 3.2 CLISTER TERMOSENSIBLE E ALTAMENTE MUCOADHESIVO COMO PORTADOR DE HUCESC-CM PARA O TRATAMENTO LOCAL DE ÚLCERAS COLORECTAIS EN EII Proponse a combinación de PF127, PF68 e MK4M como materias primas para lograr un hidroxel útil para o tratamento de úlceras na mucosa colónica, características da enfermidade inflamatoria intestinal. Os estudos realizados para acadalo devandito obxectivo preséntanse no Capítulo III e aparecen resumidos na Figura 5. Figura 5. Diagrama xeral cos ensaios descritos no Capítulo III. Coma no caso anterior, empregáronse as mesmas ferramentas de AI para estudar o efecto de cada ingrediente nas propiedades dos hidroxeis. Establecéronse os espazos de coñecemento e de deseño para producir sistemas termosensibles e altamente bioadhesivos, capaces de cargar e ceder material proteico. A xiringabilidade das mesturas ternarias está directamente relacionada coa proporción de PF127 no sistema. Ámbolos dous Pluronic® condicionan a temperatura de xelificación, a cal incrementase a medida que se incorpora PF68 no sistema. Por outra banda, o derivado celulósico contribúe á viscosidade e á bioadhesión do hidroxel. Resumo in extenso XXIX De novo, a modelaxe con redes neuronais artificiais e algoritmos xenéticos permitiu seleccionar unha formulación que presentase unha xiringabilidade adecuada (<86 mJ para unha agulla de 24Gx1.5"), unha temperatura de xelificación entre 25-27 ºC e unha bioadhesion elevada. O sistema está composto por un 16.34% de PF127, un 2.55% de PF68 e un 0.26% de MK4M. As características fisicoquímicas obtidas para a formulación optimizada resultaron ser moi similares ás preditas polo modelo, o que permitiu a súa validación. Este hidroxel empregouse como portador de medio condicionado derivado de células nai cervicais uterinas humanas (H-hUCESC-CM). Os resultados do estudo in vivo nun modelo experimental de rato con colite aguda preséntanse no capítulo IV e aparecen resumidos na Figura 6. Figura 6. Diagrama xeral cos ensaios descritos no Capítulo IV. Desta maneira, os datos experimentais indican que o tratamento co hidroxel cargado diminuiu significativamente a perda de peso corporal e o acurtamento do colon, a dexeneración da mucosa colónica e os niveis de ARNm de TNF-α, IFN-γ e IL-6. Os datos indican que o H-hUCESC-CM alivia eficazmente a colite inducida por DSS en ratos, suxerindo que o H-hUCESC-CM podería representar unha terapia sen células moi atractiva para o tratamento local da EII. LORENA GARCÍA DEL RÍO XXX 4. CONCLUSIÓNS 1. As ferramentas de IA permitiron estudar facilmente o espazo de coñecemento e establecer o espazo de deseño de formulacións ternarias para administración sublingual e rectal, incluso utilizando deseños experimentais moi reducidos. A modelaxe dos datos experimentais con estas tecnoloxías híbridas facilita extraordinariamente o deseño de hidroxeis con prestacións avanzadas. A técnica de “neurofuzzy logic” permitiu dilucidar os efectos de cada compoñente na formulación, así como as súas interaccións sobre o comportamento texturométrico, reolóxico e de liberación destes hidroxeis ternarios; adquirindo así un coñecemento profundo sobre o papel que desenrolan nos sistemas. Por outra banda, a combinación de redes neuronais artificiais e algoritmos xenéticos permitiu optimizar varias variables á vez, atopando a mellor formulación posible dentro do espazo de deseño. 2. Unha combinación adecuada de PF127, HS1200 e Gantrez® (AN119 ou S97) permitiu obter hidroxeis termosensibles e altamente mucoadhesivos con características adecuadas para a administración sublingual de moléculas proteicas (un xel cunha alta resistencia, mucoadhesión e cohesión e, rápida liberación de fármaco). Ademais, o HS1200 mostrou capacidade inmunoestimulante, potencialmente útil como adxuvante para o desenvolvemento de vacinas. O hidroxel optimizado acadado para a administración da vacina CTH522 foi capaz de estenderse sobre a mucosa sublingual, previr a fuga da proteína recombinante por inxestión ou dilución na saliva e, inducir a súa liberación de forma rápida para garantir unha absorción completa a través do tecido da mucosa oral. A formulación cargada coa vacina CTH522 probada en ratos CBF61 femia, mostrou unha efectividade dependente da estratexia de inmunización. Os resultados demostraron que a inmunización combinada de reforzo parenteralsublingual foi capaz de provocar unha resposta de IgA local no tracto xenital Resumo in extenso XXXI e mellorar de forma significativa a produción de IFN-γ polos ganglios linfáticos cervicais en comparación coa inmunización parenteral con liposomas CAF01. 3. Combinacións adecuadas de PF127, PF68 e MK4M son útiles para preparar clisteres que experimentan unha rápida transición de estado líquido a sólido cando alcanzan a temperatura corporal, minimizando a fuga e/ou a dilución das moléculas terapéuticas. Estas fórmulas intelixentes exhibiron unha boa bioadhesividade e unha rápida liberación do fármaco, e están deseñadas para promover un efecto inmediato sobre a mucosa intestinal danada. Desenvolveuse un hidroxel optmizado de caracterísiticas desexadas que mostrou unha xiringabilidade, bioadhesión e temperatura de xelificación adecuadas. Os resultados nun modelo de colite aguda en rato mostraron que esta formulación optimizada, cargada con medio condicionado de células nai uterinas cervicais humanas podería representar unha terapia atractiva sen necesidade de empregar células para o tratamento local das úlceras colorectais presentes na enfermidade inflamatoria intestinal. Expanded Abstract XXXIX 1. INTRODUCTION Mucosal administration of active molecules, such as proteins, is an attractive and advantageous approach to induce local or systemic therapeutic and/or immunostimulatory responses. However, absorption processes through mucous membranes present important limitations associated with their nature, whose structures are specially designed to constitute the physical barriers between the organism and the external environment. Critical aspects such as rapid protein drainage and enzymeor pH-mediated degradation limit the residence time of therapeutic molecules on mucosal surfaces and condition the passage of substances through the mucosa and therefore, their usefulness [1-5]. Hydrogels are 3D polymeric networks very useful as mucosal dosage forms. Their characteristics, which are highly tunable depending on their composition, can be modulated to achieve highly mucoadhesive formulations, capable of interacting with mucous membranes and increasing the residence time of drugs at the target site, enhancing their absorption and the desired therapeutic effect [68]. The possibility of preparing hydrogels by gentle methods makes them particularly attractive for the formulation of drugs with low stability, such as proteins [9-14]. In this sense, thermosensitive and mucoadhesive hydrogels are of particular interest to improve the efficacy and bioavailability of these drugs when administered on mucous membranes. Their liquid nature when administered facilitates their complete spreading and interaction with mucous membranes. After administration, their sol-gel transition at physiological temperature reduces runoffs or dilution in body fluids, favouring drug absorption and guaranteeing the maintenance of the formulation in the area until all dose is absorbed [15-17]. In addition, these hydrogels are excellent candidates to overcome the limitations of transmucosal delivery due to their biocompatibility, mucoadhesion, thermosensitivity and drug loading capacity [6, 18-21]. However, the design of such dosage forms involves the preparation of multicomponent systems, which is a challenging task in the field of pharmaceutical technology. According to the concept of quality by design (QbD), for the rational design of these systems it is necessary LORENA GARCÍA DEL RÍO XL to understand and control the effect of each raw material, the interactions between them, and to reach a compromise solution to formulate hydrogels with desired characteristics [22]. In recent years, artificial intelligence (AI) tools such as neural networks, fuzzy logic or genetic algorithms have proven to be very useful in studying the effects of composition and operational variables on the characteristics of different dosage forms such as tablets, hydrogels, nanoparticles or microparticles [22-26]. 2. OBJECTIVES This thesis work addresses the usefulness of AI tools in the development of multicomponent hydrogels with advanced performance. Thus, we explore the potential of ternary combinations of selected polymers to generate thermosensitive and highly bioadhesive hydrogels for protein loading and delivery with two applications; sublingual vaccination and local treatment of colorectal ulcers in inflammatory bowel disease (IBD). Objective I: Design of ternary, thermosensitive and mucoadhesive hydrogels as protein carriers for sublingual vaccination. A selection of polymers will be proposed, their suitability and influence on texturometric, rheological and drug release properties will be studied. In addition, the composition of the hydrogel will be optimized using AI tools and, the usefulness of the optimized hydrogel as a Chlamydia trachomatis vaccine carrier to elicit systemic and/or mucosal immune responses in mice will be evaluated. The results related to this objective are shown in chapters I and II of this report. Objective II: Design and characterization of an optimized, thermosensitive and highly mucoadhesive enema useful as a human cervical stem cell conditioned medium carrier for the local treatment of colorectal ulcers in IBD. Polymer selection will be proposed and the design space of ternary hydrogel formulations with suitable characteristics (thermosensitivity, mucoadhesivity, extensibility and retention in the rectal tract) will be established using AI tools. Then, a formulation will be optimized and the in vivo regenerative and antiinflammatory capacity of the optimized hydrogel, loaded with human cervical stem cell conditioned medium, will be evaluated in an Expanded Abstract XLI experimental model of acute colitis in mice. The results related to this objective are shown in chapters III and IV of this thesis. 2. MATERIALS AND METHODS 2.1 HYDROGELS DEVELOPMENT Pluronic® F127 (PF127), Hybrane® S1200 (HS1200) and Gantrez® varieties AN119 (AN119) and S97 (S97) were selected for the design of sublingual hydrogels, whereas Pluronic® F127 (PF127) and F68 (PF68) and the cellulosic derivative Methocel® K4M (MK4M) for the rectal enema. For the selected components to develop hydrogels for sublingual vaccination, proliferation assays were performed on human macrophages, obtained by culture and stimulation of THP-1 monocytes (ATCC®, USA), in the presence of the polymers at different concentrations. The effect on cell proliferation was assessed using the WST-1 assay kit. For hydrogels design, after the initial selection of components, a reduced experimental design was established through DataForm® v.3.1 software (Intelligensys Ltd., UK) using a balanced density method. This made possible to delimit both the knowledge space and the design space of ternary systems using a limited number of formulations. The elaboration procedure is described in detail in each case, in the corresponding section (sections 3.2.3, 3.2.4, 5.2.2 and 5.2.3 of chapters I and III). 2.2 HYDROGELS CHARACTERIZATION Texturometric analysis of hydrogels was carried out on a TA XT Plus Texture Analyzer (Surrey, UK), following previously described protocols [26-30]. This allowed the determination of syringeability parameters, gel strength, adhesion, mucoadhesion and cohesion work of hydrogels at 37 °C. In addition, an AR1000-N controlled stress rheometer (TA Instruments, UK) was used to determine the gelation temperature of the hydrogels as a function of storage (G′) and loss (G″) moduli records at different temperatures. In addition, the complex viscosity of each hydrogel was measured at 20 °C. LORENA GARCÍA DEL RÍO XLII For the evaluation of the in vitro drug release profile from hydrogels, green fluorescent antigen-like microspheres (GFM) in the case of hydrogels intended for mucosal vaccination or bovine serum albumin in the case of rectal enemas were used as “model drugs”. Loaded hydrogels were placed in 40 μm pore size release strainers and the percentage of microspheres or protein released at pre-set times was determined by the appropriate method. 2.3 HYDROGEL MODELIZATION USING ARTIFICIAL INTELLIGENCE TECHNIQUES The software FormRules® v4.03 (Intelligensys Ltd., UK), which combines artificial neural networks with fuzzy logic techniques, was used to model the hydrogel characteristics according to their composition and to analyze the effect of each material on the properties of the hydrogels using the “IF-THEN” rules it generates. In addition, INForm® v.5.01 software (Intelligensys, Ltd. UK) was used to combine artificial neural networks with genetic algorithms to model and select, in each case, an optimal formulation that gels at physiological temperature and has the best characteristics for its application and maintenance on the corresponding mucosa (resistance to washout or dilution and high mucoadhesion). The quality of the models was assessed on the basis of the determination coefficients and ANOVAs, which compare the values predicted by the model with the experimental values. The models obtained were validated by the production and characterization of optimized formulations. 2.4 EVALUATION OF THE THERAPEUTIC ACTIVITY OF DEVELOPED SYSTEMS 2.4.1 Mouse immunization assay A study was conducted in 9-week-old female CBF61 mice to evaluate the usefulness of the optimized hydrogel as a carrier for sublingual vaccines against Chlamydia trachomatis following different vaccination strategies. For this purpose, animals were divided into four groups of three to five mice. The immunization schedule is depicted in Figure 1. Groups 1 to 3 were subcutaneously Expanded Abstract XLIII primed at the base of the tail with CTH522 chlamydia vaccine formulated in CAF01 liposomes [31]. Group 4 was sublingually primed with CTH522 vaccine dispersed in the optimized hydrogel (OGEL). Then, groups 2 to 4 were boosted twice sublingually (days 15 and 35) with CTH522 alone (Group 2) or included within the optimized system (Groups 3 and 4). Group 1 received no additional doses. Figure 1. Immunization schedule and experimental groups tested. Animals were primed subcutaneously (s.c) or sublingually (s.l) using CAF01 and OGEL as vaccine delivery systems. On day 57, mice were euthanized. Total IgG and IgA CTH522specific antibodies were determined in serum and vaginal lavages at the end of the study. Additionally, IFN-γ and IL-17A concentrations were determined in spleen and cervical lymph nodes by ELISA. Differences between experimental groups were established by oneway ANOVA and Tukey’s test for multiple comparisons (p<0.05). Experiments were conducted at the Statens Serum Institut (SSI) in Denmark, in accordance with the regulations establish by the Danish Ministry of Justice and Animal Protection committees by the Danish Animal Experiments Inspection Permit 2017-15-0201-01363 and in accordance with the European Community Directive 2010/63. 2.4.1 Experimental mouse model of acute colitis An acute colitis model study was performed in male C57BL/6 mice to evaluate the usefulness of an optimized enema loaded with conditioned medium from human cervical stem cells, for the local treatment of colorectal ulcers in IBD. LORENA GARCÍA DEL RÍO XLIV To this end, the protocol described by Wirtz et al [32] was used (Figure 2). Acute colitis was induced in mice by the administration of 3.5% dextran sulphate sodium (DSS) in the drinking water for 9 days. Between 10 and 15 days, 75 μl of each treatment was administered rectally twice daily using a 26-gauge probe needle. The animals were divided into 5 groups as follows: 1) Healthy control group; 2) Untreated group: mice that have not received any treatment after DSS administration; 3) H-DMEM-F12 control group: mice treated with the hydrogel after colitis induction; 4) H-hUCESC-CM group: mice treated with the gel loaded with conditioned medium after colitis induction; and 5) Mesalazine (MZ) group: mice treated with mesalazine foam after colitis induction (standard treatment in IBD). On day 16 animals were euthanized. Figure 2. Diagram of the study design for DSS-induced colitis and parameters evaluated. Clinical symptoms (survival, changes in body weight, stool consistency and bleeding severity) were assessed and, after euthanized animals, a macroscopic examination of the colon (length) was performed. In addition, histological evaluation (degree of inflammation and cellular infiltration, microscopic ulcerations, crypt architecture, epithelial atrophy and dysplasia) and mRNA extraction from colon samples for real-time quantitative PCR determining TNFα, IL-6, and IFN-γ expression were performed. Expanded Abstract XLV The study was performed according to the protocol approved by the Ethics Committee for Animal Experiments of the University of Santiago de Compostela. 3. RESULTS AND DISCUSSION 3.1 THERMOSENSITIVE AND MUCOADHESIVE TERNARY HYDROGELS AS PROTEIN CARRIERS FOR SUBLINGUAL IMMUNIZATION First, the combination of PF127, HS1200 and Gantrez® (AN119 or S97) is proposed to produce a hydrogel for sublingual application, useful for mucosal immunization. The studies carried out for this purpose are presented in chapter I and summarized in Figure 3. Figure 3. General diagram of assays performed on Chapter I. Studies on macrophages indicated a potential immunostimulatory effect of HS1200 by promoting the proliferation of these antigenpresenting cells. On the other hand, no toxicity was observed for any of the polymers. AI tools, in particular the combination of artificial neural networks and fuzzy logic (neurofuzzy logic) allowed to establish the role of each polymer in the rheological and texturometric behaviour of hydrogels, as well as its capacity to retain microspheres used as model antigen (GFM). Thus, the interaction between PF127 and Gantrez® rules gelation temperature, mucoadhesion and adhesion work and, gel strength. Differences were observed between the two varieties of Gantrez® evaluated, with the S97 variety allowing a slower microspheres release. The incorporation of HS1200 in the LORENA GARCÍA DEL RÍO XLVI formulation resulted in a reduction in gel strength and adhesion capacity. The combination of artificial neural networks and genetic algorithms was used to model the composition of hydrogels and to optimize the process and thus select the ternary composition that provides systems with maximum mucoadhesion, cohesion and gel strength, and which also gel at body temperature. The studies carried out for this purpose are presented in chapter II and summarized in Figure 4. Figure 4. General diagram of assays performed on Chapter II. The optimal composition selected by the model generated by the INForm® software (21.1% PF127, 5% HS1200 and 0.08% Gantrez® AN119) was elaborated and characterized. Additionally, it was stored under controlled conditions to evaluate its stability. The rheological and texturometric characteristics of the elaborated hydrogels are in complete agreement with those predicted by the model, which allowed its validation. Furthermore, the formulation was found to be stable. The potential usefulness of this optimized hydrogel as sublingual carrier of CTH522 Chlamydia trachomatis vaccine was evaluated in a mouse model. The results of the study showed that the most promising strategy for obtaining local genital tract immunity was the combination of parenteral priming with CTH522 included in Expanded Abstract XLVII liposomes, followed by sublingual boosting with the CTH522 vaccine-loaded optimized hydrogel, which achieved the best immune responses. The thermosensitive properties of the hydrogel facilitate its application under the tongue and increased contact with the sublingual tissue. The gelation at body temperature together with the fast release of antigen must prevent the loss of CTH522 vaccine by swallowing and promote its absorption. In vivo studies demonstrated that parenteral-sublingual boost immunization, using CAF01 and the optimized hydrogel as carriers of CTH522 vaccine, was more effective in inducing cell-mediated immune responses (Th1/Th17) than mucosal or parenteral vaccination alone. In addition, this strategy resulted in an increase in local production of IgA in the genital tract. 3.2 THERMOSENSITIVE AND HIGHLY BIOADHESIVE ENEMA AS CARRIER OF H-UCESC-CM FOR LOCAL TREATMENT OF COLORECTAL ULCERS IN IBD A combination of PF127, PF68 and MK4M is proposed as raw materials to achieve a hydrogel useful in the treatment of rectal and colonic mucosal ulcers, which are characteristic of inflammatory bowel disease. The studies carried out towards this goal are presented in chapter III and summarized in Figure 5. Figure 5. General scheme and assays performed on Chapter III. As previously, identical AI tools were used to study the effect of each ingredient on hydrogels properties. Both, the knowledge space LORENA GARCÍA DEL RÍO LIV [29] C.A. Ramírez Barragán, E.R. Macías Balleza, L. GarcíaUriostegui, J.A. Andrade Ortega, G. Toríz, E. Delgado, Rheological characterization of new thermosensitive hydrogels formed by chitosan, glycerophosphate, and phosphorylated β-cyclodextrin, Carbohydrate polymers, 201 (2018) 471-481. [30] M. Campana-Seoane, A. Peleteiro, R. Laguna, F.J. OteroEspinar, Bioadhesive emulsions for control release of progesterone resistant to vaginal fluids clearance, Int J Pharm, 477 (2014) 495-505. [31] C.B. Roces, S. Khadke, D. Christensen, Y. Perrie, ScaleIndependent Microfluidic Production of Cationic Liposomal Adjuvants and Development of Enhanced Lymphatic Targeting Strategies, Mol Pharm, 16 (2019) 4372-4386. [32] S. Wirtz, V. Popp, M. Kindermann, K. Gerlach, B. Weigmann, S. Fichtner-Feigl, M.F. Neurath, Chemically induced mouse models of acute and chronic intestinal inflammation, Nat Protoc, 12 (2017) 1295-1309. 1. INTRODUCTION 1.Introduction 3 1.1. MUCOSAL SURFACES 1.1.1. Anatomy and function (small overview) A mucosal surface or mucous membrane is any tissue able to secrete mucus across a layer of epithelial cells [1, 2]. Mucosal surfaces are the interface between the external and internal environment [3, 4]. They cover an extensive part of the body (≈ 400 m2), including the inner ear, eye conjunctiva, the ducts of all exocrine glands and the respiratory, gastrointestinal, and urogenital tracts [5, 6]. Mucous membranes are physical barriers for allergens, carcinogens, and infectious agents. Their selective permeability allows the nutrients and air uptake, as well as the flux of ions, solutes and water. Furthermore, they are also involved in other survival functions such as perception of signals and reproduction [3, 4, 7]. As mucosal surfaces are daily exposed to different environmental hazards, they are endowed with powerful chemical and mechanical defence mechanisms. Coating with a mucous network is one of the strategies of mucosal surfaces for protecting living cells from external agents [8]. The secretion of mucus is essential to shield epithelial cells against enzymatic, chemical, microbial and mechanical challenges [2, 9, 10]. Moreover, a large and highly specialized mucosal immune system ensures their protection developing innate and adaptive responses against potential threats [6, 11]. Higher mammals have evolved a sophisticated defence system associated to mucosal surfaces to: a) protect the body from invasion of viruses, bacteria, fungi and parasites; b) remove or isolate non-microbial environmental substances and; c) destroy potential cancer cells that emerge in the body, also known as immune surveillance [1, 12, 13]. Thereby, the mucosal immune system is a specific immune organization that protects mucosal compartments via articulating immune responses through the immune cells residing in them. In addition, the mucosal immune system has many unusual and unique features such as a distinctive anatomy, specialized mechanisms for antigen uptake, as well as, regulatory and effector responses to induce tolerance against food and innocuous antigens [11, 14]. LORENA GARCÍA DEL RÍO 4 In healthy human adults, the mucosal immune system comprises almost 80% of all lymphocytes, being the largest immune tissue of the body [15]. These lymphocytes can be accumulated in, or migrate between various mucosal-associated lymphoid tissues (MALT). MALT principal function is to produce and secrete immunoglobulins A (IgA) across mucosae via antigen specific, Th1, Th2 or cytotoxic Tcell mediated responses [16]. MALT functions independently of the systemic immune system and it is highly compartmentalized [6]. The best-known MALT representatives are: nasopharynx associated lymphoid tissue (NALT), bronchus-associated lymphoid tissue (BALT), gut-associated lymphoid tissue (GALT) and genital associated lymphoid tissue (Figure 1). However, the lacrimal ductassociated (LDALT), the conjunctiva-associated (CALT), the larynxassociated (LALT) and salivary duct-associated (DALT) lymphoid tissues have also been described [16, 17]. Although MALT compartments have similar histology and features, their immunobiology and histogenesis could differ depending on the location [17]. Figure 1. Overall scheme of mucosa-associated lymphoid tissue (MALT) and its most important subcompartments: nasopharynx associated lymphoid tissue 1.Introduction 5 (NALT), bronchus-associated lymphoid tissue (BALT), gut-associated lymphoid tissue (GALT) and the genital lymphoid associated tissue. Reproduced from Nils Lycke (2012) with permission of Springer Nature. 1.1.2. Mucus and mucins Mucus is an adhesive and viscoelastic complex hydrogel secreted by specialized Goblet and mucous cells located in the gastrointestinal and reproductive tracts, airways and the eyes. Mucus at the oral mucosa is secreted as part of saliva by the minor and major salivary glands [18-20]. It is continuously produced, discharged onto mucosal surfaces, and finally recycled, digested, or discarded, allowing surfaces removal of bacteria and debris [8, 18]. Mucus includes salts, nucleic acids, lipids, cells, cellular debris, antimicrobial molecules, secreted antibodies and a variety of proteins, being mucins the most representative. Mucins are glycoproteins with abundant serine, threonine and proline amino acid residues, which are characterized by threadlike core protein domains rich in O-linked oligosaccharides, giving rise to their characteristic “bottle brush” conformation. The high content of sialic acid and sulfate groups in their backbone confers a strong negative charge to mucins, which increases mucus stiffness through charge repulsion, giving gel-like properties [2, 10, 18, 21]. Mucus network has a high number of physical entanglements via covalent and non-covalent interactions, including electrostatic, hydrophobic, hydrogen bonds and also specific binding interactions. These interactions contribute to the viscoelasticity of the mucus, creating a mesh with a pore size that varies between 100 and 2000 nm depending on the mucosal tissue location [18, 21]. The properties of the mucosal barriers depend on the composition, the degree of glycosylation and conformation of the mucins. The tissue of origin and environmental factors (ionic strength, pH and the presence of other agents) determine the structure of the glycoproteins, and therefore, the thickness of the mucus and its functions [2, 22]. Mucus layer can difficult the holding of drug carriers on mucosal surfaces if prepared with non-mucoadhesive materials. These materials can interact with mucus glycoproteins through different chemical and physical mechanisms increasing the residence time of LORENA GARCÍA DEL RÍO 6 delivery systems on mucosal surfaces and therefore, enhancing absorption, bioavailability and efficacy of the drugs [23-27]. 1.2. INMUNITY FROM MOUTH TO RECTUM 1.2.1. Oral cavity immune system The first compartment of the gastrointestinal tract is the oral cavity. Anatomically, the oral mucosa is located between the skin of the face and the mucosa lining of the gastrointestinal tract, sharing features with both tissues and the respiratory tract [14, 28, 29]. In the oral cavity there are keratinized (hard palate, gingiva and outer lips) and non-keratinized regions (ventral side of the tongue, the floor of the mouth and the inner surface of the lips and cheeks), the latter being more elastic and permeable for antigens or drug delivery [20, 29, 30]. Oral epithelium is stratified and made up of three layers: basal, intermediate and superficial, that act as a mechanical barrier protecting the subjacent tissues against environmental hazards or fluid loss. Beneath oral epithelium are the basement membrane, the lamina propria and the submucosa (Figure 2) [20, 29]. Oral tissue contains different immune cells such as lymphoid cells, macrophage-like cells, antigen-presenting cells (APCs) and some pro-inflammatory cells [31]. Langerhans cells (LCs), located in the suprabasal layer, are the most superficial APCs, meanwhile the myeloid and plasmacytoid dendritic cells subsets are in the lamina propria and submucosal tissue, respectively [29]. Little is known about the functioning of oral mucosa immune system. However, two theoretical models have been proposed: 1) The presence of different oral-pharyngeal domains comprising the MALT as: buccal mucosa (inner cheeks), salivary glands, Waldeyer's ring (consisted of palatine tonsils and adenoids) and pharyngeal lymphoid tissues [14, 32]. 2) The presence of inductive and effector sites, resembling the gastrointestinal immune system: oral inductive sites as Waldeyer's ring, lymphoid follicles and proximal draining lymph nodes (submaxillary, internal jugular and superficial 1.Introduction 7 cervical), and effector sites as the epithelium, the lamina propria and salivary glands [14, 28, 33]. Figure 2. Structure of oral pharyngeal cavity and scheme of immunization process. Antigens delivered on the oral mucosa are taken up by Langerhans cells in the epithelial layer and also by myeloid dendritic cells and macrophages in the lamina propria. Antigen-bearing dendritic cells migrate to secondary lymphoid tissue and lymph nodes to induce adaptive immune responses. MALTs of the oral-pharyngeal cavity are located in the tonsils, where micro-fold cells transfer antigens to antigen-presenting cells. Lastly, oral mucosa is moistened by salivary glands, that produce mucin and saliva. Saliva contains several antimicrobial peptides and proteins such as peroxidases, lysozymes, defensins, lactoferrin, and immunoglobulins (e.g. secretory IgA). Their functions are lubrication of tissues, swallowing, prevention of tooth demineralization and dental protection [20, 29, 34]. 1.2.2. Gut-associated lymphoid tissues or GALT The intestinal mucosa is the largest surface of the body, in close contact with commensal microbiota, food and pathogens [35]. In the defence of the intestinal mucosa participate the intestinal epithelial LORENA GARCÍA DEL RÍO 8 barrier, the lamina propria, the GALT and the draining lymph nodes [36]. Specifically, the GALT and the duodenopancreatic, para-aortic and mesenteric lymph nodes are the main locations for priming adaptive immune cell responses in the gut [37]. The intestinal mucosa covers the small intestine, the caecum and the colon. The intestinal epithelial barrier is composed by a single layer of columnar epithelial cells arranged in crypts and villi, designed for nutrient absorption. Villi are not present in the caecum and colon [36, 37]. This epithelium is constantly renewed by immature cells arising from the base of the crypts. It is protected by substances such as mucins and antimicrobial peptides (AMPs), synthesized by Goblet and Paneth cells, respectively [4, 36]. Thus, the apical surface of intestinal epithelial cells (IEC) is covered by a protective mucus layer composed mainly by mucins [4]. Furthermore, in this mucous layer there is also IgA secreted by enterocytes [38]. The lamina propria is made up by loosely packed connective tissue. Different innate immune cells (e.g. macrophages, dendritic cells (DCs) and innate lymphoid cells) and adaptive lymphocytes (e.g. IgA-producing plasma cells and effector T cells) can be found diffusively in the lamina propria. This set of cells is called the immune effector site [38]. Furthermore, the lamina propria has other functions such as blood and nervous supply and lymph drainage [37]. The GALT is formed by organized subepithelial lymphoid follicles located in the intestinal mucosa and submucosa (Figure 3) [37] and characterized by an overlying follicle-associated epithelium (FAE) that contains microfold cells (M cells). M cells are a specialized subset of intestinal epithelial cells responsible for the uptake and transport of antigens from the lumen to the subepithelial dome (SED) region, which has plenty of dendritic cells (DCs) that deliver antigens to adaptive immune cells [37, 38]. When lymphoid follicles aggregate, they form macroscopic structures known as Peyer’s, caecal and colonic patches. Specifically, Peyer’s patches are oval-shaped aggregates of hundreds of B cells lymphoid follicles that are flanked by smaller T cell regions [37, 38]. Peyer’s patches, predominant in the distal ileum, are considered the main inductive site for the mucosal immune response. 1.Introduction 9 Figure 3. Scheme of intestinal mucosa and immune responses. Reproduced from Ahluwalia et al. (2017) with permission of Taylor & Francis. Lymph nodes are also important immune organs that contain large numbers of lymphocytes housed in their paracortex, cortex and medulla. Unlike Peyer’s patches, lymph nodes are encapsulated lymphoid tissue and have afferent lymphatics [36]. Mesenteric lymph nodes can filter the lymph coming from the gut and also initiate immune responses against free antigens or delivered by DCs from the lamina propria. The first line of defence to protect the host against harmful agents are the cells of the innate immune system, which include macrophages, DCs, neutrophils, natural killer cells, eosinophils, mast cells and basophils. When DCs and other phagocytic cells of intestinal mucosa interact with pathogenic microorganisms, LORENA GARCÍA DEL RÍO 16 Thereby, several dosage forms for sublingual application including multi-layered nanofibrous mucoadhesive films, freeze drying formulations, nanoand microparticles, thermosensitive hydrogels and liposomes have been developed to protect and enhance antigen absorption and thus increase B and T cell immunogenicity [52, 81, 8688]. In addition to confer immunity against harmful pathogens, sublingual delivery of antigens is also used in immunotherapy to induce tolerance against allergens such as pollen and food allergens [89]. On the other hand, despite buccal delivery of antigens has been less explored than sublingual one, some studies revealed that buccal route could also generate potent cell-mediated immune responses [9092]. Very encouraging results have been obtained after buccal immunization with a vaccine based on bacteriophage MS2-L2 viruslike particles encoding epitopes of different human papillomavirus (HPV) adjuvanted with cholera toxin and monophosporyl lipid A. This approach effectively induced protective immunity in both genital and oral regions against eleven oncogenic HPV types associated with a 95% and a 99% of cervical and head and neck cancers, respectively [91]. In addition, the therapeutic potential of a HPV-16 synthetic long peptide has been evaluated in a buccal tumor model. Interestingly, intratumoral administration of this vaccine without any adjuvant increased systemic and local antigen-specific CD8+ T cells, leading to a potent antitumor effect against HPV-associated lesions presented in buccal mucosa [92]. 1.3.2.4. Oral vaccination Oral administration of antigens is highly desirable because it is patient-friendly, cost-effective, and easy to use. However, the development of effective oral vaccine formulations is extremely challenging owing the intrinsic features of gastrointestinal tract [93]. Curiously, except FluMistTM (a nasal spray flu vaccine), the few licensed mucosal vaccines available are administrated by oral route and are inactivated or live attenuated vaccines against Vibrio cholerae, Salmonella thyphimurium, adenovirus (for military use only) and poliovirus [46]. Although a vast research has been made to find effective non-living oral vaccines against these and other enteric 1.Introduction 17 pathogens, the efforts have not yet borne fruit. Lipid based delivery systems, including liposomes, ISCOM®s and emulsions; virus-like particles, microand nanoparticles, pH-responsive hydrogels, tablets and capsules have been proposed as vaccine delivery platforms to enhance enteric mucosal immune responses [94-98]. In addition to enteric infections, oral mucosa has been also evaluated to confer immunity against other diseases such as hepatitis, tuberculosis, chlamydia and HIV [96, 98-100]. 1.3.2.5. Vaginal and rectal vaccination Basically, intravaginal immunization is used generally to induce immunity against genital pathogens such as HIV, HSV and chlamydia. Although rectal mucosa has also been explored and evaluated for this purpose, vaginal antigen administration is a more common approach [101-106]. Intravaginal immunization is able to elicit specific-systemic and mucosal immune responses, promote local IgG and IgA secretion antibodies as well as IgG secretion. Vaginallyadministered vaccine strategies include the use of live attenuated and DNA vaccines, as well as, delivery systems comprising liposome based gel formulations, lyophilized solid dosage formulations, viral vectors, peptide-based carriers or vaginal ring devices [106-112]. Intravaginal vaccination was also carried out as a successful method to treat HPV-associated cervical cancer and increased survival in mice. A therapeutic approach included submucosal vaginal immunization with the pcDNA3-CRT/E7 DNA vaccine encoding IL-2 following by electroporation. Other strategy was to administer carboplatin/paclitaxel chemotherapy prior to intravaginal vaccination using a HPV16-E7 synthetic long peptide adjuvanted with CpG (cytosine-guanine oligodeoxynucleotide) [113, 114]. 1.4. GASTROINTESTINAL INFLAMMATORY MUCOSAL PATHOLOGIES Inflammatory Bowel Disease (IBD) encloses a group of inflammatory disorders that affect the gastrointestinal tract [115, 116]. They are chronic, systemic, complex, and multifactorial pathologies in which patients suffer from relapsing and flares accompanied with stomach and abdominal pain, severe diarrhoea, weight loss, cramping and rectal bleeding [117, 118]. LORENA GARCÍA DEL RÍO 18 Ulcerative colitis (UC) and Crohn’s Disease (CD) are the most representative gastrointestinal inflammatory disorders. Although CD and UC share clinical features such as mucosal injury, periods of relapsing and remitting mucosal inflammation, as well as a decrease in the mucus layer thickness and its spread depending on the severity of inflammation, their pathology is different [115, 119-121]. CD can discontinuously affect any region of the digestive tract, being the distal ileum and colon the most commonly damaged areas. It involves the entire intestine wall, from mucosa to serosa, implying a transmural inflammatory process which frequently lead to the development of perianal fistulas [115, 119, 122, 123]. On the other hand, UC is restricted to the large intestine where the mucosa and submucosa are uniformly affected. Starting in the rectum, the inflammation could spread to the whole colon. However, in some rare cases the ileum could also be harmed [115, 124-126]. Besides, their immunological basis are also well differentiated, whereas in CD predominate Th1 responses, in UC the overexpressed cytokines are Th2 type [123, 127]. Several theories have been proposed in order to elucidate the origin of IBD. Autoimmunity, the infection with microbes such as Mycobacterium avium subsp. paratuberculosis or Escherichia coli among others, dysbiosis in commensalism bacteria, together with genetic and environmental factors were attributed as potential causes for IBD development [127-132]. In addition, it is interesting to hallmark that IBD exists mainly in Western countries and, the incidence rate is continuously rising also in emerging countries [132, 133]. It seems that intestinal mucosal barrier dysfunction and immune dysregulation play a significant role in the development, progression and perpetuation of IBD [134-136]. IBD is related to augmented permeability in both non-ulcerated and ulcerated epithelia of the gastrointestinal tract, strongly suggesting that this defect in mucosal barrier function precedes gut inflammation [137, 138]. There are alterations in several components of the epithelium and its adhesion molecules. For example, some studies have shown decreased expression of tight junctions as well as changes in their function, 1.Introduction 19 ultrastructure and protein composition during disease outbreaks. Multiple alterations in mucins expression and mucus composition together with a decreased number and depletion of Globet cells lead to a reduction in the thickness of the mucus layer, which can sometimes be disrupted. In addition, defects in defensin and AMPs production, as well as in pattern recognition receptors and low secretion of total IgA by B cells have also been linked with IBD. Finally, increased epithelial apoptosis in both UC and CD also contributes to barrier leakiness [2, 135, 136, 138]. Collectively, these alterations in mucosal epithelial barrier causes loss of fluid and solutes, leading to leak flux diarrhoea together with increased antigen translocation. Some studies suggested that inflammatory response is initiated due to an aberrant local immune response against commensal microbiota [2, 134, 135, 139]. Thus, the increased permeability of intestinal epithelium, owing to defects in Ncadherins synthesis, allows the entry of commensal flora into the lamina propria, triggering pro-inflammatory cytokine responses such as TNF-α, IL-6 and IL-1β that initiate and maintain gut inflammation [2, 134, 139]. It has also been suggested that DCs and macrophages could be related to the exacerbated immune response in IBD. Macrophages contribute to CD pathogenesis owing to their capability to produce pro-inflammatory cytokines such as IL-12 and IL-23, and to activate T cells. In addition, disorders in phagocyte function of macrophages have frequently been associated to chronic colitis similar to CD. On the other hand, DCs populations in patients suffering from IBD are highly activated, producing high levels of proinflammatory cytokines. Besides, any dysregulation that affects one or more DCs subsets functions could result in a shift from regulatory to pathogenic T cells, exacerbating gut inflammation [2, 137]. However, despite many efforts, IBD aetiology remains unclear, but data obtained from different studies revealed that genetic, immune and environmental factors are mainly involved. Hence, meanwhile IBD origin is not completely elucidated, the main therapeutic goal is to regain epithelial barrier homeostasis and thus achieve mucosal healing and clinical remission. The process of intestinal mucosal healing is very complex and not fully understood. What is known, LORENA GARCÍA DEL RÍO 20 however, is that restoring barrier integrity through rapid wound closure is crucial to limit antigen translocation and loss of fluids and electrolytes. Unfortunately, there are not therapies available that restore and regenerate epithelial barrier function, but some emerging and promising approaches such as immunotherapy, faecal microbiota transplantation, probiotics, antibiotics with immune-regulating properties like ciprofloxacin or metronidazole and cell therapies or its derivatives, among others, have been proposed [135, 138, 140, 141]. 1.5. HYDROGELS: TYPES AND PROPERTIES Hydrogels are translucent or transparent soft semisolid materials traditionally used in medicine, cosmetics, pharmacy or materials science. Recently, gels have attracted extraordinary attention for their advantageous properties in a wide variety of applications [142-145]. As delivery platforms they can protect the drug from extreme pH or enzyme degradation, improving its stability [142, 146]. The minimum hydrogel composition includes the liquid medium (or solvent), and a gelling agent (polymer). Small proportion of a crosslinker must be also necessary [147]. Gelling agents, form a three-dimensional (3D) colloidal network structure able of immobilizing and trapping solvent molecules, limiting fluid flow. The solvent can be found as free solvent, entrapped within the network or bound as a solvation layer. For hydrogels the solvent is always water [145]. Hydrogels are widely used in the pharmaceutical field due to their similarity to biological tissues and their biocompatibility. Their matrix, similar to extracellular matrices, have interstitial spaces capable of containing large amounts of water, while maintaining their structure and resistance to dilution due to physical or chemical crosslinking [148-150]. The aqueous medium gives them high permeability to oxygen and allows the diffusion of nutrients and small molecules. Therefore, hydrogels are extremely attractive platforms as superabsorbent materials, tissue engineering scaffolds, contact lenses, biosensors, and drug delivery systems [86, 151]. Despite their valuable advantages, hydrogels also present some limitations associated to high polymer crystallinity, low solubility, unfavourable thermal and mechanical properties, unreacted monomers, material- 1.Introduction 21 dependent biodegradability and the employment of toxic crosslinkers [152]. Hydrogels are produced by the reaction of one or more polymer/monomer/cross-linker units. These small or large organic bridging molecules or particles generate twoand three-dimensional aggregates of various structured types, which through crosslinking and/or hierarchical self-assembly procedures give rise to a 3D hydrocolloidal network [144, 147, 153, 154]. The gelling process is determined by polymer-solvent and polymer-polymer interactions. An increase in the concentration of the polymer reduces the distance between bridging particles, which leads to crosslinking and entanglements. As a result, the immobilization of the components and an increase in the mechanical and viscoelastic properties of the system takes place [145]. The mechanical strength and the hydrogels diffusion properties are determined by the interactions between the aqueous solvent and the polymeric networks and by the nature of the bridging molecules, their shape, molecular size, ionic nature and degree of hydrophilicity [147, 153]. Hydrogels are capable of increasing more than 20% of their weight in contact with water or biological fluids, due to the presence of polar hydrophilic groups in their polymer chain, -COOH, -OH, - NH2, -CONH2 and -SO3H [153, 155, 156]. The hydrogel structure does not collapse during swelling or dissolve in solution due to their reticulated meshwork [152, 157]. Thus, cross-linking provides the sufficient physical integrity and mechanical strength to maintain hydrogel 3D structure in the swollen state [153]. Cross-linking could take place during the preparation of the hydrogel or in vivo, once applied in the human body [152]. Hydrogel cross-linking can be achieved either chemically or physically. Chemical cross-linking involves the formation of new covalent bonds by reaction of functional groups between the different polymer chains with or without a cross-linking agent [152, 153, 158]. Physically cross-linked hydrogels or supramolecular hydrogels are also known as reversible hydrogels [142, 152]. Amphiphilic polymers are widely used to produce this type of systems. Physical LORENA GARCÍA DEL RÍO 22 cross-linking does not require the use of cross-linking agents, being based on non-covalent interactions such as hydrogen bonding, dipoledipole, van der Waals, π-π stacking or charge transfer that form nonpermanent reticulation nodes between the short hydrophobic sequences present in the long hydrophilic polymer chains [142, 144]. Physical hydrogels frequently respond to changes in the environment, as pH, temperature or ionic strength, leading to reversible sol-gel transitions. The absence of external cross-linkers is beneficial to maintain low toxicity and biocompatibility and also to allow their post process bulk modification and reshaping [152, 158]. Thus, this type of hydrogels has received considerable attention during the last decade due to their tuneable properties and versatility [142]. Most of the hydrogel properties, such as the shear modulus (G), the swelling ratio (Q) or the diffusion coefficient of entrapped molecules (D), are controlled by the polymer concentration and the cross-linking density (ρx; number of physical or chemical cross-links by volume) (Figure 4) [159]. Cross-linking density, determines the pores or meshes of the network (ξ), and therefore, the maximum molecular size that can pass through the hydrogel matrix, controlling drug diffusivity [156, 159-161]. Figure 4. Two network structures presenting low and high cross-linking densities are shown to demonstrate the relationship between cross-linking 1.Introduction 23 density and equilibrium swelling ratio (Q), diffusion coefficient (D) and shear modulus (G). As the cross-linking density increases, the space available between the polymer chains or mesh size (ξ) decreases, reducing the diffusion of solutes. Reprinted with permission from Kirschner and Anseth, 2013. Generally, the mesh size reported range from 5 to 100 nm. Most hydrogels possess a broad distribution of mesh sizes due to polymer polydispersity and network heterogeneity. Other factors such as the selected polymer or external stimuli (pH and temperature) also can have an effect on hydrogel mesh size [156]. Mesh size is crucial when using hydrogels as drug delivery systems (Figure 5). If the drug is smaller than the mesh (rmesh/rdrug >1), the network will not limit the mobility of the bioactive compound and the drug release process will occur mainly by diffusion. If the mesh is similar in size to the drug (rmesh/rdrug ≈ 1), the steric hindrance effect becomes prominent. The polymer chains restrict drug diffusion, and an extended drug release profile can be obtained. Finally, when the mesh size is extremely small relative to the drug molecules (rmesh/rdrug <1), the strong steric hindrance prevents their mobility, resulting in their physically entrapment in the hydrogel matrix (reservoir), from which it can only be released by hydrogel degradation, mesh size enlargement by a swelling process or network deformation due to a mechanical force, a magnetic-field or ultrasounds [156, 162]. The release behaviour of hydrogels is also affected by other drug molecular configuration (e.g. charge, shape and deformability) [161]. Therefore, drug release kinetics from hydrogels is controlled by mechanisms of diffusion, swelling and/or degradation of the system as shown in Figure 5 [161, 162]. Hydrogels are versatile and tunable platforms for drug delivery, whose release profiles can be tailored through a selection of suitable polymer or combination of polymers (variety, molecular weight, concentration), and crosslinking density [155, 160]. LORENA GARCÍA DEL RÍO 24 Figure 5. Drug diffusion is mediated by mesh size. Reprinted with permission from Li and Mooney, 2016. 1.5.1. Stimuli-sensitive hydrogels Stimuli-sensitive hydrogels are smart hydrogels formed by polymers including stimuli-responsive moieties, enabling structural changes, triggered by an external stimulus such as pH, temperature, ionic strength, electrical and magnetic fields, light, ultrasound irradiation and redox processes. Stimuli-sensitive hydrogels can be classified as physically, chemically and biochemically responsive hydrogels [151, 152]. 1.5.1.1. Physically responsive hydrogels Thermosensitive hydrogels Thermosensitive hydrogels are systems produced by polymers with hydrophobic and hydrophilic components in their structure. They are sensitive to temperature changes in the surrounding environment, which drastically affects their morphology 1.Introduction 25 and can lead to a physical phase transition from solution to the gel state [163]. Their response to temperature can be negative or positive. Positive responsive hydrogels as polyacrylamide and poly(acrylic acid)-based hydrogels, swell at temperatures over their upper critical solution temperature (UCST). Below the UCST, they shrink due to the formation of a complex structure through hydrogen bonding, which leads to network dehydration by releasing fluids or solvents from its matrix [155, 164]. Negative responsive hydrogels, as for example polyvinylpyrrolidon/poly(N-isopropylacrilamide), are characterized for swelling below their low critical solution temperature (LCST) due to hydrogen bonds formation between water and the hydrophilic part of the network. Whereas at a temperature above LCST, hydrophobic interactions predominate, weakening interchain hydrogen bonds and leading to the shrinkage of the hydrogel [165]. Another mechanism of temperature-dependent gel formation was observed for amphiphilic triblock copolymers composed of hydrophilic polyethylene oxide (PEO) and hydrophobic polypropylene oxide (PPO), commonly known as Pluronic® or Poloxamers. Above their critical micelle concentration (CMC), unassociated unimers of these copolymers equilibrate with micelles formed by PPO blocks in the inner part of micelles and a corona of PEO segments that interact with water. However, micelle formation strongly depends on temperature. The heating of the polymer solution to the LCST, usually designed as critical micelle temperature (CMT), promotes micelle formation as PPO chains become less soluble leading to micelle packing and gelation. Pluronic® are widely used for in situ gelling hydrogels characterized by their safety, bioadhesiveness, stability and ability to form gels at low concentrations at body temperature. Pluronic® gelation properties can be adjusted combining different Pluronic® or other excipients [166, 167]. In situ gelling hydrogels suffer a sol-gel transition under physiological conditions. As a consequence of this transition hydrogels are formed on the site of injection allowing easy drug loading, complete filling of the defect/wound and easy to handling, being suitable for intramuscular, subcutaneous, topical, transdermal LORENA GARCÍA DEL RÍO 32 mucoadhesiveness. At lower concentrations, the interaction between the polymer and the mucus is unstable due to the reduced number of penetrating polymer chains per volume of mucus. While at higher concentrations, the coiling of the polymer chains occurs, and the accessibility of the solvent and the interpenetration of polymer/mucin chains are reduced, worsening the mucoadhesive properties [220]. On the other hand, crosslinking degree affects mucoadhesive properties by controlling polymer chain mobility. Highly cross-linked materials are capable of swell in contact with biological fluids and retain water in their structure, resisting dissolution, but their mucoadhesiveness is reduced by decreasing the mobility of the polymer chains [222]. Polymer mucoadhesion is also affected by the magnitude of its hydration, which must be adequate to expand and create a macromolecular mesh that allows the polymer-mucin interactions to be established. When the polymer swells, its mucoadhesive areas are exposed, allowing its mechanical entanglement with the mucosal network. If excessive hydration occurs, mucoadhesion is reduced due to the formation of a slippery mucilage [219, 220, 222]. Since the properties of hydrogels are determined by the ingredients chosen for their preparation, an adequate selection of polymers together with a proper understanding of how selected materials interact with each other is crucial to obtain hydrogels of desired characteristics. Therefore, obtaining formulations that meet all the requirements for successful mucosal drug release can be feasible through proper hydrogel design, which should allow determining the optimal polymer concentration to achieve highly mucoadhesive formulations capable of overcoming intrinsic barriers present in mucosal tissues. 1.6. ARTIFICIAL INTELLIGENCE TECHNIQUES APPLIED TO PHARMACEUTICAL SYSTEMS DESIGN The design and manufacture of multicomponent pharmaceutical systems is challenging as it involves a large number of variables whose single effects or in combination have a great impact on the final properties of the dosage forms [223-225]. The quality of pharmaceutical systems is directly related to the knowledge of the 1.Introduction 33 effects of these variables on the characteristics of the formulations (knowledge space) and the production of the systems within the limits of the design space. Knowledge space and design space, make up the concept of Quality by Design (QbD), the systematic approach defined by the International Conference on Harmonization for pharmaceutical development (ICH Q8 guideline). The concept of QbD was first introduced into the pharmaceutical industry in 2004 as a consequence of the Food and Drug Administration (FDA) initiative named ‟cGMPs for the 21th century: A Risk-Based Approach”. Then, European and Japanese regulatory authorities have also encouraged their use in Chemistry, Manufacturing and Controls review process. Thus, QbD has emerged as an approach to promote a better understanding of pharmaceutical products, from the early stages (design and development) to subsequent commercial production [226, 227]. Nevertheless, despite the advantages that QbD offers to pharmaceutical companies, it is not completely implemented on real product development or industrial applications [223, 227]. QbD points out the need to identify critical parameters that affect the quality, efficacy, stability and safety of pharmaceutical dosage forms which is extremely difficult in multivariate processes using traditional methodology. When the number of variables is large, the numerical solutions for a process optimization is time consuming and entered the realm of the complex statistics [228, 229]. In recent years, the growing development and introduction of Artificial Intelligence (AI) tools comes to help in the task of designing complex systems, including pharmaceutical formulations. AI is used to provide accurate analysis, interpretation and management of generated data throughout the development and production of a product, making easier to overcome potential setbacks as well as save time and money [223, 230]. Machine learning, a subset of AI, is the most widely used AI technology in pharmaceutical development. These computerized techniques include Artificial Neural Networks (ANNs), genetic algorithms and neurofuzzy logic [225, 231]. ANNs are the main tools for data mining. ANNs are mathematical models generated by computational systems, designed to mimic the biological processes of the human brain. Like biological neural LORENA GARCÍA DEL RÍO 34 systems, ANNs are made up of unitary elements called perceptrons, or nodes, organized in layers and forming complex networks (Figure 6). Perceptrons receive one or more input from neighbouring neurons, process the information and produce an output to be transmitted to the next node. The strengths of connections between the artificial neurons are called ‟weights” [226]. ANNs is a supervised AI technique. During the training process, the network structure, weights, threshold, and transfer function must be established in order to find an algorithm that relates inputs and outputs of the training set. Thus, ANN learns and is able to answer “WHAT-IF” questions which can be used to predict results of unrealized experiences and identify complex and latent patterns between experimental data (input) and response values (output) (Figure 6). ANNs can detect intricate non-linear relationships within big datasets by creating nonlinear input-output mappings. For this reason, ANNs arouse as a powerful and robust technique for modelling, predicting, and optimizing pharmaceutical processes and formulations, underpinning the application of QbD in pharma industry [225, 227, 232-236]. The interpretation of ANNs is quite difficult due to the generation of black-box models. For that reason, ANNs can be combined with other techniques to facilitate their interpretation and/or utility. Its combination with fuzzy logic lead hybrid technologies named neurofuzzy logic systems. Fuzzy logic is a form of logic in which the truth value of a variable can be expressed with a word and a real number between 0 and 1, inclusive, called the degree of membership. Fuzzification and defuzzification processes allow encoding the structure of ANNs into a reasonably small number of logical rules. In addition to predictive models, neurofuzzy logic provides easy-tofollow rules generated in the explicit “IF-THEN” format (Figure 6), representing the output-input relationships within the database. This makes cause-and-effect relationships between inputs and outputs easier to understand and therefore, allow to define the knowledge space and the design space [223, 224, 234, 237]. 1.Introduction 35 Figure 6. Typical diagram of an artificial neural network, which consists of unitary elements called perceptrons or nodes. The main structural components of an artificial neural network are input, hidden and output layers. The input layer transfer information to the hidden layer. This layer consists of several interconeccted nodes that link the input and output layers via weights (w). After the training process, the inputs and outputs are linked by an algorithm that allows answering the “WHAT-IF” questions and thus predicting the outcomes of the unrealized experiences. ANNs can also be combined with genetic algorithms (GA). GA are also bioinspired AI tools. Based on the biological principles of genetic variation and natural selection, genetic algorithms are designed to select the best solution of a specific problem. When combined ANN and GA, a set of candidate solutions to the problem are generated and selected according to their fitness to previous established criteria. After several generations, GA are able to find a global optimal solution for different attributes simultaneously [238]. Overall, the combination of ANNs with neurofuzzy logic or genetic algorithms is a feasible and promising approach to design drug delivery systems. It allows to precisely control how procedural variables affect the final properties of pharmaceutical formulations as well as the further obtaining of optimized dosage forms with suitable characteristics. LORENA GARCÍA DEL RÍO 36 REFERENCES [1] A.E. Williams, Immunology: Mucosal and Body Surface Defences, Wiley, 2011. [2] J. Mestecky, W. Strober, M.W. Russell, H. Cheroutre, N. Lambrecht, B.L. Kelsall, Mucosal Immunology, Elsevier Science, 2015. [3] J.H. Rhee, Current and New Approaches for Mucosal Vaccine Delivery, in: Mucosal Vaccines, 2020, pp. 325-356. [4] M.M. France, J.R. Turner, The mucosal barrier at a glance, J Cell Sci, 130 (2017) 307-314. [5] S.H. Kim, Y.-S. Jang, The development of mucosal vaccines for both mucosal and systemic immune induction and the roles played by adjuvants, Clinical and Experimental Vaccine Research, 6 (2017). [6] J. Holmgren, C. Czerkinsky, Mucosal immunity and vaccines, Nat Med, 11 (2005) S45-53. [7] P. Brandtzaeg, Mucosal immunity: induction, dissemination, and effector functions, Scand J Immunol, 70 (2009) 505-515. [8] G.C. Hansson, Mucus and mucins in diseases of the intestinal and respiratory tracts, J Intern Med, 285 (2019) 479-490. [9] S.K. Linden, P. Sutton, N.G. Karlsson, V. Korolik, M.A. McGuckin, Mucins in the mucosal barrier to infection, Mucosal Immunol, 1 (2008) 183-197. [10] J. Witten, T. Samad, K. Ribbeck, Selective permeability of mucus barriers, Curr Opin Biotechnol, 52 (2018) 124-133. [11] K.M. Murphy, C. Weaver, Janeway’s Immunobiology: Ninth International Student Edition, Taylor & Francis Group, 2016. [12] J.R. McGhee, K. Fujihashi, Inside the mucosal immune system, PLoS Biol, 10 (2012) e1001397. [13] E.P. Widmaier, H. Raff, K.T. Strang, Vander's Human Physiology: The Mechanisms of Body Function, McGraw-Hill Education, 2018. [14] R.Q. Wu, D.F. Zhang, E. Tu, Q.M. Chen, W. Chen, The mucosal immune system in the oral cavity-an orchestra of T cell diversity, Int J Oral Sci, 6 (2014) 125-132. [15] H. Tlaskalová-Hogenová, L. Tucková, R. Lodinová-Zádniková R. Stepánková, B. Cukrowska, D.P. Funda, I. Striz, H. Kozáková, I. 1.Introduction 37 Trebichavský, D. Sokol, Z. Reháková, J. Sinkora, P. Fundová, D. Horáková, L. Jelínková, D. Sánchez, Mucosal immunity: its role in defense and allergy. [16] M.F. Cesta, Normal structure, function, and histology of mucosaassociated lymphoid tissue, Toxicol Pathol, 34 (2006) 599-608. [17] G.A. Parker, T.L. Papenfuss, Immune System, in: Atlas of Histology of the Juvenile Rat, 2016, pp. 293-347. [18] J. Leal, H.D.C. Smyth, D. Ghosh, Physicochemical properties of mucus and their impact on transmucosal drug delivery, Int J Pharm, 532 (2017) 555-572. [19] R. Bansil, B.S. Turner, The biology of mucus: Composition, synthesis and organization, Adv Drug Deliv Rev, 124 (2018) 3-15. [20] V.F. Patel, F. Liu, M.B. Brown, Advances in oral transmucosal drug delivery, J Control Release, 153 (2011) 106-116. [21] T. Pelaseyed, J.H. Bergström, J.K. Gustafsson, A. Ermund, G.M.H. Birchenough, A. Schütte, S. van der Post, F. Svensson, A.M. Rodríguez-Piñeiro, E.E.L. Nyström, C. Wising, M.E.V. Johansson, G.C. Hansson, The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system, Immunol Rev, 260 (2014) 8-20. [22] H.H. Sigurdsson, J. Kirch, C.M. Lehr, Mucus as a barrier to lipophilic drugs, Int J Pharm, 453 (2013) 56-64. [23] J.D. Smart, Theories of Mucoadhesion, (2014) 159-174. [24] N.A. Peppas, J.B. Thomas, J. McGinty, Molecular aspects of mucoadhesive carrier development for drug delivery and improved absorption, J Biomater Sci Polym Ed, 20 (2009) 1-20. [25] B.M. Boddupalli, Z.N. Mohammed, R.A. Nath, D. Banji, Mucoadhesive drug delivery system: An overview, J Adv Pharm Technol Res, 1 (2010) 381-387. [26] J. Xu, S. Strandman, J.X. Zhu, J. Barralet, M. Cerruti, Genipincrosslinked catechol-chitosan mucoadhesive hydrogels for buccal drug delivery, Biomaterials, 37 (2015) 395-404. [27] J. Xu, M. Tam, S. Samaei, S. Lerouge, J. Barralet, M.M. Stevenson, M. Cerruti, Mucoadhesive chitosan hydrogels as rectal drug delivery vessels to treat ulcerative colitis, Acta Biomater, 48 (2017) 247-257. LORENA GARCÍA DEL RÍO 38 [28] M. Ana Valentich, T. Analia Cafaro, H. Marcelo Serra, Oral Cavity-Associated Immune System: What is New?, Current Immunology Reviews, 7 (2011) 253-263. [29] H. Kraan, H. Vrieling, C. Czerkinsky, W. Jiskoot, G. Kersten, J.P. Amorij, Buccal and sublingual vaccine delivery, J Control Release, 190 (2014) 580-592. [30] N.M. Moutsopoulos, J.E. Konkel, Tissue-Specific Immunity at the Oral Mucosal Barrier, Trends Immunol, 39 (2018) 276-287. [31] P. Moingeon, Update on immune mechanisms associated with sublingual immunotherapy: practical implications for the clinician, J Allergy Clin Immunol Pract, 1 (2013) 228-241. [32] F.X. Lu, R.S. Jacobson, Oral mucosal immunity and HIV/SIV infection, J Dent Res, 86 (2007) 216-226. [33] C.W. Cutler, R. Jotwani, Dendritic cells at the oral mucosal interface, J Dent Res, 85 (2006) 678-689. [34] M. Naafs, Oral Mucosal Immune Suppression, Tolerance and Silencing: A Mini-Review, Modern Approaches in Dentistry and Oral Health Care, 1 (2018). [35] M. Flach, A. Diefenbach, Development of Gut-Associated Lymphoid Tissues, in: Mucosal Immunology, 2015, pp. 31-42. [36] B. Ahluwalia, M.K. Magnusson, L. Ohman, Mucosal immune system of the gastrointestinal tract: maintaining balance between the good and the bad, Scand J Gastroenterol, 52 (2017) 1185-1193. [37] A.M. Mowat, W.W. Agace, Regional specialization within the intestinal immune system, Nat Rev Immunol, 14 (2014) 667-685. [38] H. Ohno, Intestinal M cells, J Biochem, 159 (2016) 151-160. [39] M.R. Neutra, N.J. Mantis, J.P. Kraehenbuhl, Collaboration of epithelial cells with organized mucosal lymphoid tissues, Nat Immunol, 2 (2001) 1004-1009. [40] A. Srivastava, D.V. Gowda, S.V. Madhunapantula, C.G. Shinde, M. Iyer, Mucosal vaccines: a paradigm shift in the development of mucosal adjuvants and delivery vehicles, APMIS, 123 (2015) 275288. [41] M. Li, Y. Wang, Y. Sun, H. Cui, S.J. Zhu, H.J. Qiu, Mucosal vaccines: Strategies and challenges, Immunol Lett, 217 (2020) 116125. 1.Introduction 39 [42] M.R. Neutra, P.A. Kozlowski, Mucosal vaccines: the promise and the challenge, Nat Rev Immunol, 6 (2006) 148-158. [43] K.A. Woodrow, K.M. Bennett, D.D. Lo, Mucosal vaccine design and delivery, Annu Rev Biomed Eng, 14 (2012) 17-46. [44] C. Hervouet, C. Luci, N. Cuburu, M. Cremel, S. Bekri, L. Vimeux, C. Maranon, C. Czerkinsky, A. Hosmalin, F. Anjuere, Sublingual immunization with an HIV subunit vaccine induces antibodies and cytotoxic T cells in the mouse female genital tract, Vaccine, 28 (2010) 5582-5590. [45] M.T. De Magistris, Mucosal delivery of vaccine antigens and its advantages in pediatrics, Adv Drug Deliv Rev, 58 (2006) 52-67. [46] A. Miquel-Clopes, E.G. Bentley, J.P. Stewart, S.R. Carding, Mucosal vaccines and technology, Clin Exp Immunol, 196 (2019) 205-214. [47] D. Christensen, Vaccine adjuvants: Why and how, Hum Vaccin Immunother, 12 (2016) 2709-2711. [48] S. Apostolico Jde, V.A. Lunardelli, F.C. Coirada, S.B. Boscardin, D.S. Rosa, Adjuvants: Classification, Modus Operandi, and Licensing, J Immunol Res, 2016 (2016) 1459394. [49] J.F. Mann, R. Acevedo Campo, J.D Campo, O. Pérez, V.A. Ferro, Delivery systems: a vaccine strategy for overcoming mucosal tolerance?, Expert Rev Vaccines, 8 (2009) 103-112. [50] H.X. Tan, A.K. Wheatley, R. Esterbauer, S. Jegaskanda, J.J. Glass, D. Masopust, R. De Rose, S.J. Kent, Induction of vaginalresident HIV-specific CD8 T cells with mucosal prime-boost immunization, Mucosal Immunol, 11 (2018) 994-1007. [51] A. Sato, A. Suwanto, M. Okabe, S. Sato, T. Nochi, T. Imai, N. Koyanagi, J. Kunisawa, Y. Kawaguchi, H. Kiyono, Vaginal memory T cells induced by intranasal vaccination are critical for protective T cell recruitment and prevention of genital HSV-2 disease, J Virol, 88 (2014) 13699-13708. [52] J.A. White, J.S. Blum, N.A. Hosken, J.O. Marshak, L. Duncan, C. Zhu, E.B. Norton, J.D. Clements, D.M. Koelle, D. Chen, W.C. Weldon, M.S. Oberste, M. Lal, Serum and mucosal antibody responses to inactivated polio vaccine after sublingual immunization LORENA GARCÍA DEL RÍO 40 using a thermoresponsive gel delivery system, Hum Vaccin Immunother, 10 (2014) 3611-3621. [53] J.S. Woodworth, D. Christensen, J.P. Cassidy, E.M. Agger, R. Mortensen, P. Andersen, Mucosal boosting of H56:CAF01 immunization promotes lung-localized T cells and an accelerated pulmonary response to Mycobacterium tuberculosis infection without enhancing vaccine protection, Mucosal Immunol, 12 (2019) 816-826. [54] P. Ralli-Jain, D. Tifrea, C. Cheng, S. Pal, L.M. de la Maza, Enhancement of the protective efficacy of a Chlamydia trachomatis recombinant vaccine by combining systemic and mucosal routes for immunization, Vaccine, 28 (2010) 7659-7666. [55] Y. Fujkuyama, D. Tokuhara, K. Kataoka, R.S. Gilbert, J.R. McGhee, Y. Yuki, H. Kiyono, K. Fujihashi, Novel vaccine development strategies for inducing mucosal immunity, Expert Rev Vaccines, 11 (2012) 367-379. [56] N. Marasini, L.M. Kaminskas, Subunit-based mucosal vaccine delivery systems for pulmonary delivery - Are they feasible?, Drug Dev Ind Pharm, 45 (2019) 882-894. [57] A.B. Nesburn, I. Bettahi, X. Zhang, X. Zhu, W. Chamberlain, R.E. Afifi, S.L. Wechsler, L. Benmohamed, Topical/Mucosal Delivery of Sub-Unit Vaccines That Stimulate the Ocular Mucosal Immune System, The Ocular Surface, 4 (2006) 178-187. [58] R. Da Costa Martins, C. Gamazo, M. Sanchez-Martinez, M. Barberan, I. Penuelas, J.M. Irache, Conjunctival vaccination against Brucella ovis in mice with mannosylated nanoparticles, J Control Release, 162 (2012) 553-560. [59] M. Sánchez-Martínez, R. da Costa Martins, G. Quincoces, C. Gamazo, C. Caicedo, J.M Irache, I. Peñuelas, Radiolabeling and biodistribution studies of polymeric nanoparticles as adjuvants for ocular vaccination against brucellosis, Rev Esp Med Nucl Imagen Mol, 32 (2013) 92-97. [60] S. Yoon, E.D. Kim, M.S. Song, S.J. Han, T.K. Park, K.S. Choi, Y.K. Choi, K.Y. Seo, Eyedrop Vaccination Induced Systemic and Mucosal Immunity against Influenza Virus in Ferrets, PLoS One, 11 (2016) e0157634. 1.Introduction 41 [61] M.C. Moran, A.R. Bence, M.F.S. Vallecillo, C.M. Lutzelschwab, M.G. Rodriguez, R. Pardo, F.A. Goldbaum, V. Zylberman, S.D. Palma, B.A. Maletto, S.M. Estein, Polymeric antigen BLSOmp31 formulated with class B CpG-ODN in a nanostructure (BLSOmp31/CpG-ODN/Coa-ASC16) administered by parenteral or mucosal routes confers protection against Brucella ovis in Balb/c mice, Res Vet Sci, 135 (2021) 217-227. [62] A. Inic-Kanada, M. Stojanovic, S. Schlacher, E. Stein, S. BelijRammerstorfer, E. Marinkovic, I. Lukic, J. Montanaro, N. Schuerer, N. Bintner, V. Kovacevic-Jovanovic, O. Krnjaja, U.B. Mayr, W. Lubitz, T. Barisani-Asenbauer, Delivery of a Chlamydial Adhesin NPmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers, PLoS One, 10 (2015) e0144380. [63] A.B. Nesburn, S. Slanina, R.L.Burke, H. Ghiasi, S. Bahri, S.L. Wechsler, Local periocular vaccination protects against eye disease more effectively than systemic vaccination following primary ocular herpes simplex virus infection in rabbits, J Virol, 72 (1998) 77157721. [64] A.B. Nesburn, T.V. Ramos, X. Zhu, H. Asgarzadeh, V. Nguyen, L. BenMohamed, Local and systemic B cell and Th1 responses induced following ocular mucosal delivery of multiple epitopes of herpes simplex virus type 1 glycoprotein D together with cytosinephosphate-guanine adjuvant, Vaccine, 23 (2005) 873-883. [65] T.N. Alkie, A. Yitbarek, K. Taha-Abdelaziz, J. Astill, S. Sharif, Characterization of immunogenicity of avian influenza antigens encapsulated in PLGA nanoparticles following mucosal and subcutaneous delivery in chickens, PLoS One, 13 (2018) e0206324. [66] M. Wu, H. Zhao, M. Li, Y. Yue, S. Xiong, W. Xu, Intranasal Vaccination with Mannosylated Chitosan Formulated DNA Vaccine Enables Robust IgA and Cellular Response Induction in the Lungs of Mice and Improves Protection against Pulmonary Mycobacterial Challenge, Front Cell Infect Microbiol, 7 (2017) 445. [67] R. Nakahashi-Ouchida, Y. Yuki, H. Kiyono, Cationic pullulan nanogel as a safe and effective nasal vaccine delivery system for respiratory infectious diseases, Hum Vaccin Immunother, 14 (2018) 2189-2193. LORENA GARCÍA DEL RÍO 48 [119] N. Gobbato, M. Rachid, G. Perdigon, Anti-inflammatory effect of yoghurt in an experimental inflammatory bowel disease in mouse, J Dairy Res, 75 (2008) 497-504. [120] X.Y. Bai, Y. Yan, L. Wang, L.G. Zhao, K. Wang, Novel pHsensitive hydrogels for 5-aminosalicylic acid colon targeting delivery: in vivo study with ulcerative colitis targeting therapy in mice, Drug Deliv, 23 (2016) 1926-1932. [121] J. Zhou, M. O'Keeffe, G. Liao, F. Zhao, C. Terhorst, B. Xu, Design and Synthesis of Nanofibers of Self-assembled de novo Glycoconjugates towards Mucosal Lining Restoration and AntiInflammatory Drug Delivery, Tetrahedron, 72 (2016) 6078-6083. [122] P.L. Kozuch, S.B. Hanauer, Treatment of inflammatory bowel disease: a review of medical therapy, World J Gastroenterol, 14 (2008) 354-377. [123] L. Steidler, Microbiological and immunological strategies for treatment of inflammatory bowel disease, Microbes Infect, 3 (2001) 1157-1166. [124] A.K. Dash, Z. Gong, D.W. Miller, H. Huai-Yan, J. Laforet, Development of a rectal nicotine delivery system for the treatment of ulcerative colitis, Int J Pharm, 190 (1999) 21-34. [125] J. Meier, A. Sturm, Current treatment of ulcerative colitis, World J Gastroenterol, 17 (2011) 3204-3212. [126] T.J. Keane, J. Dziki, E. Sobieski, A. Smoulder, A. Castleton, N. Turner, L.J. White, S.F. Badylak, Restoring Mucosal Barrier Function and Modifying Macrophage Phenotype with an Extracellular Matrix Hydrogel: Potential Therapy for Ulcerative Colitis, J Crohns Colitis, 11 (2017) 360-368. [127] C.N. Bernstein, J.D. Forbes, Gut Microbiome in Inflammatory Bowel Disease and Other Chronic Immune-Mediated Inflammatory Diseases, Inflamm Intest Dis, 2 (2017) 116-123. [128] J.T. Kuenstner, S. Naser, W. Chamberlin, T. Borody, D.Y. Graham, A. McNees, J. Hermon-Taylor, A. Hermon-Taylor, C.T. Dow, W. Thayer, J. Biesecker, M.T. Collins, L.A. Sechi, S.V. Singh, P. Zhang, I. Shafran, S. Weg, G. Telega, R. Rothstein, H. Oken, S. Schimpff, H. Bach, T. Bull, I. Grant, J. Ellingson, H. Dahmen, J. Lipton, S. Gupta, K. Chaubey, M. Singh, P. Agarwal, A. Kumar, J. 1.Introduction 49 Misri, J. Sohal, K. Dhama, Z. Hemati, W. Davis, M. Hier, J. Aitken, E. Pierce, N. Parrish, N. Goldberg, M. Kali, S. Bendre, G. Agrawal, R. Baldassano, P. Linn, R.W. Sweeney, M. Fecteau, C. Hofstaedter, R. Potula, O. Timofeeva, S. Geier, K. John, N. Zayanni, H.M. Malaty, C. Kahlenborn, A. Kravitz, A. Bulfon, G. Daskalopoulos, H. Mitchell, B. Neilan, V. Timms, D. Cossu, G. Mameli, P. Angermeier, T. Jelic, R. Goethe, R.A. Juste, L. Kuenstner, The Consensus from the Mycobacterium avium ssp. paratuberculosis (MAP) Conference 2017, Front Public Health, 5 (2017) 208. [129] A. Watad, S. Azrielant, N.L. Bragazzi, K. Sharif, P. David, I. Katz, G. Aljadeff, M. Quaresma, G. Tanay, M. Adawi, H. Amital, Y. Shoenfeld, Seasonality and autoimmune diseases: The contribution of the four seasons to the mosaic of autoimmunity, J Autoimmun, 82 (2017) 13-30. [130] T. Azimi, M.J. Nasiri, A.S. Chirani, R. Pouriran, H. Dabiri, The role of bacteria in the inflammatory bowel disease development: a narrative review, APMIS, 126 (2018) 275-283. [131] H. Ebrahimiyan, S. Aslani, N. Rezaei, A. Jamshidi, M. Mahmoudi, Survivin and autoimmunity; the ins and outs, Immunol Lett, 193 (2018) 14-24. [132] G. Rogler, L. Biedermann, M. Scharl, New insights into the pathophysiology of inflammatory bowel disease: microbiota, epigenetics and common signalling pathways, Swiss Med Wkly, 148 (2018) w14599. [133] S.C. Ng, G.G. Kaplan, W. Tang, R. Banerjee, B. Adigopula, F.E. Underwood, D. Tanyingoh, S.C. Wei, W.C. Lin, H.H. Lin, J. Li, S. Bell, O. Niewiadomski, M.A. Kamm, Z. Zeng, M. Chen, P. Hu, D. Ong, C.J. Ooi, K.L. Ling, Y. Miao, J. Miao, H.J. de Silva, M. Niriella, S. Aniwan, J. Limsrivilai, P. Pisespongsa, K. Wu, H. Yang, K.K. Ng, H.H. Yu, Y. Wang, Q. Ouyang, M. Abdullah, M. Simadibrata, J. Gunawan, I. Hilmi, K.L. Goh, Q. Cao, H. Sheng, A. Ong-Go, V.H. Chong, J.Y.L. Ching, J.C.Y. Wu, F.K.L. Chan, J.J.Y. Sung, Population Density and Risk of Inflammatory Bowel Disease: A Prospective Population-Based Study in 13 Countries or Regions in Asia-Pacific, Am J Gastroenterol, (2018). LORENA GARCÍA DEL RÍO 50 [134] M. Vasovic, N. Gajovic, D. Brajkovic, M. Jovanovic, N. Zdravkovaic, T. Kanjevac, The relationship between the immune system and oral manifestations of inflammatory bowel disease: a review, Cent Eur J Immunol, 41 (2016) 302-310. [135] F.E.O. Holmberg, J. Pedersen, P. Jørgensen, C. Soendergaard, K.B. Jensen, O.H. Nielsen, Intestinal barrier integrity and inflammatory bowel disease: Stem cell‐based approaches to regenerate the barrier, Journal of Tissue Engineering and Regenerative Medicine, 12 (2017) 923-935. [136] E. Martini, S.M. Krug, B. Siegmund, M.F. Neurath, C. Becker, Mend Your Fences: The Epithelial Barrier and its Relationship With Mucosal Immunity in Inflammatory Bowel Disease, Cell Mol Gastroenterol Hepatol, 4 (2017) 33-46. [137] F. Sanchez de Medina, I. Romero-Calvo, C. Mascaraque, O. Martinez-Augustin, Intestinal inflammation and mucosal barrier function, Inflamm Bowel Dis, 20 (2014) 2394-2404. [138] M.A. Odenwald, J.R. Turner, The intestinal epithelial barrier: a therapeutic target?, Nature Reviews Gastroenterology & Hepatology, 14 (2016) 9-21. [139] I. Koboziev, F. Karlsson, M.B. Grisham, Gut-associated lymphoid tissue, T cell trafficking, and chronic intestinal inflammation, Ann N Y Acad Sci, 1207 Suppl 1 (2010) E86-93. [140] S. Danese, G. Roda, L. Peyrin-Biroulet, Evolving therapeutic goals in ulcerative colitis: towards disease clearance, Nat Rev Gastroenterol Hepatol, 17 (2020) 1-2. [141] S. Pouya, M. Heidari, K. Baghaei, H. Asadzadeh Aghdaei, A. Moradi, S. Namaki, M.R. Zali, S.M. Hashemi, Study the effects of mesenchymal stem cell conditioned medium injection in mouse model of acute colitis, Int Immunopharmacol, 54 (2018) 86-94. [142] J. Mayr, C. Saldias, D. Diaz Diaz, Release of small bioactive molecules from physical gels, Chem Soc Rev, 47 (2018) 1484-1515. [143] C. Echeverria, S.N. Fernandes, M.H. Godinho, J.P. Borges, P.I.P. Soares, Functional Stimuli-Responsive Gels: Hydrogels and Microgels, Gels, 4 (2018). 1.Introduction 51 [144] C.M. Papadakis, C. Tsitsilianis, Responsive Hydrogels from Associative Block Copolymers: Physical Gelling through Polyion Complexation, Gels, 3 (2017). [145] M. Justin-Temu, F. Damian, R. Kinget, G.Van Den Mooter, Intravaginal gels as drug delivery systems, J Womens Health (Larchmt), 13 (2004) 834-844. [146] L. Mao, Y. Lu, M. Cui, S. Miao, Y. Gao, Design of gel structures in water and oil phases for improved delivery of bioactive food ingredients, Crit Rev Food Sci Nutr, 60 (2020) 1651-1666. [147] M.J. Taylor, P. Tomlins, T.S. Sahota, Thermoresponsive Gels, Gels, 3 (2017). [148] J. Hurler, A. Engesland, B. Poorahmary Kermany, N. ŠkalkoBasnet, Improved texture analysis for hydrogel characterization: Gel cohesiveness, adhesiveness, and hardness, Journal of Applied Polymer Science, 125 (2012) 180-188. [149] A. Sosnik, K.P. Seremeta, Polymeric Hydrogels as Technology Platform for Drug Delivery Applications, Gels, 3 (2017). [150] S. Chatterjee, P.C. Hui, C.W. Kan, Thermoresponsive Hydrogels and Their Biomedical Applications: Special Insight into Their Applications in Textile Based Transdermal Therapy, Polymers (Basel), 10 (2018). [151] M. Vazquez-Gonzalez, I. Willner, Stimuli-Responsive Biomolecule-Based Hydrogels and Their Applications, Angew Chem Int Ed Engl, (2019). [152] F. Ullah, M.B. Othman, F. Javed, Z. Ahmad, H. Md Akil, Classification, processing and application of hydrogels: A review, Mater Sci Eng C Mater Biol Appl, 57 (2015) 414-433. [153] K. Varaprasad, G.M. Raghavendra, T. Jayaramudu, M.M. Yallapu, R. Sadiku, A mini review on hydrogels classification and recent developments in miscellaneous applications, Mater Sci Eng C Mater Biol Appl, 79 (2017) 958-971. [154] H. Li, N. Kong, B. Laver, J. Liu, Hydrogels Constructed from Engineered Proteins, Small, 12 (2016) 973-987. [155] F. Raza, H. Zafar, Y. Zhu, Y. Ren, A. Ullah, A.U. Khan, X. He, H. Han, M. Aquib, K.O. Boakye-Yiadom, L. Ge, A Review on Recent LORENA GARCÍA DEL RÍO 52 Advances in Stabilizing Peptides/Proteins upon Fabrication in Hydrogels from Biodegradable Polymers, Pharmaceutics, 10 (2018). [156] J. Li, D.J. Mooney, Designing hydrogels for controlled drug delivery, Nat Rev Mater, 1 (2016). [157] S. Bashir, M. Hina, J. Iqbal, A.H. Rajpar, M.A. Mujtaba, N.A. Alghamdi, S. Wageh, K. Ramesh, S. Ramesh, Fundamental Concepts of Hydrogels: Synthesis, Properties, and Their Applications, Polymers (Basel), 12 (2020). [158] A. Ali, S. Ahmed, Recent Advances in Edible Polymer Based Hydrogels as a Sustainable Alternative to Conventional Polymers, J Agric Food Chem, 66 (2018) 6940-6967. [159] C.M. Kirschner, K.S. Anseth, Hydrogels in Healthcare: From Static to Dynamic Material Microenvironments, Acta Mater, 61 (2013) 931-944. [160] A. Chyzy, M. Tomczykowa, M.E. Plonska-Brzezinska, Hydrogels as Potential Nano-, Microand Macro-Scale Systems for Controlled Drug Delivery, Materials (Basel), 13 (2020). [161] S. Kirchhof, M. Abrami, V. Messmann, N. Hammer, A.M. Goepferich, M. Grassi, F.P. Brandl, Diels-Alder Hydrogels for Controlled Antibody Release: Correlation between Mesh Size and Release Rate, Mol Pharm, 12 (2015) 3358-3368. [162] D. Rocha-García, A. Guerra-Contreras, S. Rosales-Mendoza, G. Palestino, Role of porous silicon/hydrogel composites on drug delivery, Open Material Sciences, 3 (2016). [163] Q. Shi, H. Liu, D. Tang, Y. Li, X. Li, F. Xu, Bioactuators based on stimulus-responsive hydrogels and their emerging biomedical applications, NPG Asia Materials, 11 (2019). [164] S. Graham, P.F. Marina, A. Blencowe, Thermoresponsive polysaccharides and their thermoreversible physical hydrogel networks, Carbohydr Polym, 207 (2019) 143-159. [165] C. Alvarez-Lorenzo, V.Y. Grinberg, T.V. Burova, A. Concheiro, Stimuli-sensitive cross-linked hydrogels as drug delivery systems: Impact of the drug on the responsiveness, Int J Pharm, 579 (2020) 119157. 1.Introduction 53 [166] F. Zahir-Jouzdani, J.D. Wolf, F. Atyabi, A. Bernkop-Schnürch, In situ gelling and mucoadhesive polymers: why do they need each other?, Expert Opinion on Drug Delivery, 15 (2018) 1007-1019. [167] B. Shriky, A. Kelly, M. Isreb, M. Babenko, N. Mahmoudi, S. Rogers, O. Shebanova, T. Snow, T. Gough, Pluronic F127 thermosensitive injectable smart hydrogels for controlled drug delivery system development, Journal of Colloid and Interface Science, 565 (2020) 119-130. [168] L. Mude, B.K.R. Sanapalli, A.N. V, S.K. Singh, V. Karri, Overview of in situ gelling injectable hydrogels for diabetic wounds, Drug development research, (2021). [169] M.R. Matanovic, J. Kristl, P.A. Grabnar, Thermoresponsive polymers: insights into decisive hydrogel characteristics, mechanisms of gelation, and promising biomedical applications, Int J Pharm, 472 (2014) 262-275. [170] M. Norouzi, B. Nazari, D.W. Miller, Injectable hydrogel-based drug delivery systems for local cancer therapy, Drug Discov Today, 21 (2016) 1835-1849. [171] S.M. Rossi, B.K. Ryan, H.M. Kelly, Evaluation of the activity of a chemo-ablative, thermoresponsive hydrogel in a murine xenograft model of lung cancer, Br J Cancer, 123 (2020) 369-377. [172] W.K. Bae, M.S. Park, J.H. Lee, J.E. Hwang, H.J. Shim, S.H. Cho, D.E. Kim, H.M. Ko, C.S. Cho, I.K. Park, I.J. Chung, Docetaxelloaded thermoresponsive conjugated linoleic acid-incorporated poloxamer hydrogel for the suppression of peritoneal metastasis of gastric cancer, Biomaterials, 34 (2013) 1433-1441. [173] S.M. Rossi, T.E. Murray, J. Cassidy, M.J. Lee, H.M. Kelly, A Custom Radiopaque Thermoresponsive Chemotherapy-Loaded Hydrogel for Intratumoural Injection: An In Vitro and Ex Vivo Assessment of Imaging Characteristics and Material Properties, Cardiovasc Intervent Radiol, 42 (2019) 289-297. [174] R. Li, Y. Li, Y. Wu, Y. Zhao, H. Chen, Y. Yuan, K. Xu, H. Zhang, Y. Lu, J. Wang, X. Li, X. Jia, J. Xiao, Heparin-Poloxamer Thermosensitive Hydrogel Loaded with bFGF and NGF Enhances Peripheral Nerve Regeneration in Diabetic Rats, Biomaterials, 168 (2018) 24-37. LORENA GARCÍA DEL RÍO 54 [175] D.F. Argenta, B.D.C. Bernardo, A.F. Chamorro, P.R. Matos, T. Caon, Thermosensitive hydrogels for vaginal delivery of secnidazole as an approach to overcome the systemic side-effects of oral preparations, Eur J Pharm Sci, 159 (2021) 105722. [176] L. Li, J.M. Scheiger, P.A. Levkin, Design and Applications of Photoresponsive Hydrogels, Adv Mater, 31 (2019) e1807333. [177] I. Tomatsu, K. Peng, A. Kros, Photoresponsive hydrogels for biomedical applications, Adv Drug Deliv Rev, 63 (2011) 1257-1266. [178] T.L. Rapp, C.A. DeForest, Targeting drug delivery with light: A highly focused approach, Adv Drug Deliv Rev, 171 (2021) 94-107. [179] M.E. Roth-Konforti, M. Comune, M. Halperin-Sternfeld, I. Grigoriants, D. Shabat, L. Adler-Abramovich, UV Light-Responsive Peptide-Based Supramolecular Hydrogel for Controlled Drug Delivery, Macromol Rapid Commun, 39 (2018) e1800588. [180] J. Shang, Z. Shao, X. Chen, Electrical behavior of a natural polyelectrolyte hydrogel: chitosan/carboxymethylcellulose hydrogel, Biomacromolecules, 9 (2008) 1208-1213. [181] X. Ying, Y. Wang, J. Liang, J. Yue, C. Xu, L. Lu, Z. Xu, J. Gao, Y. Du, Z. Chen, Angiopep-conjugated electro-responsive hydrogel nanoparticles: therapeutic potential for epilepsy, Angew Chem Int Ed Engl, 53 (2014) 12436-12440. [182] J.H. Ha, H.H. Shin, H.W. Choi, J.H. Lim, S.J. Mo, C.D. Ahrberg, J.M. Lee, B.G. Chung, Electro-responsive hydrogel-based microfluidic actuator platform for photothermal therapy, Lab Chip, 20 (2020) 3354-3364. [183] N. Vigues, F. Pujol-Vila, A. Marquez-Maqueda, X. MunozBerbel, J. Mas, Electro-addressable conductive alginate hydrogel for bacterial trapping and general toxicity determination, Anal Chim Acta, 1036 (2018) 115-120. [184] I. Carayon, A. Gaubert, Y. Mousli, B. Philippe, Electroresponsive hydrogels: macromolecular and supramolecular approaches in the biomedical field, Biomater Sci, 8 (2020) 5589-5600. [185] I. Lee, C.W. Park, S.S. Yoon, H.M. Yang, Facile synthesis of copper ferrocyanide-embedded magnetic hydrogel beads for the enhanced removal of cesium from water, Chemosphere, 224 (2019) 776-785. 1.Introduction 55 [186] Q. Liu, H. Li, K.Y. Lam, Optimization of Deformable Magnetic-Sensitive Hydrogel-Based Targeting System in Suspension Fluid for Site-Specific Drug Delivery, Mol Pharm, 15 (2018) 46324642. [187] A. Najafipour, A. Gharieh, A. Fassihi, H. Sadeghi-Aliabadi, A.R. Mahdavian, MTX-Loaded Dual Thermoresponsive and pHResponsive Magnetic Hydrogel Nanocomposite Particles for Combined Controlled Drug Delivery and Hyperthermia Therapy of Cancer, Mol Pharm, 18 (2021) 275-284. [188] M. Noh, Y.H. Choi, Y.H. An, D. Tahk, S. Cho, J.W. Yoon, N.L. Jeon, T.H. Park, J. Kim, N.S. Hwang, Magnetic NanoparticleEmbedded Hydrogel Sheet with a Groove Pattern for Wound Healing Application, ACS Biomater Sci Eng, 5 (2019) 3909-3921. [189] X. Chen, M. Fan, H. Tan, B. Ren, G. Yuan, Y. Jia, J. Li, D. Xiong, X. Xing, X. Niu, X. Hu, Magnetic and self-healing chitosanalginate hydrogel encapsulated gelatin microspheres via covalent cross-linking for drug delivery, Mater Sci Eng C Mater Biol Appl, 101 (2019) 619-629. [190] W. Xue, X.L. Liu, H. Ma, W. Xie, S. Huang, H. Wen, G. Jing, L. Zhao, X.J. Liang, H.M. Fan, AMF responsive DOX-loaded magnetic microspheres: transmembrane drug release mechanism and multimodality postsurgical treatment of breast cancer, J Mater Chem B, 6 (2018) 2289-2303. [191] S. Kondaveeti, A.T.S. Semeano, D.R. Cornejo, H. Ulrich, D.F.S. Petri, Magnetic hydrogels for levodopa release and cell stimulation triggered by external magnetic field, Colloids Surf B Biointerfaces, 167 (2018) 415-424. [192] H. Wu, L. Song, L. Chen, Y. Huang, Y. Wu, F. Zang, Y. An, H. Lyu, M. Ma, J. Chen, N. Gu, Y. Zhang, Injectable thermosensitive magnetic nanoemulsion hydrogel for multimodal-imaging-guided accurate thermoablative cancer therapy, Nanoscale, 9 (2017) 1617516182. [193] R.V. Ulijn, N. Bibi, V. Jayawarna, P.D. Thornton, S.J. Todd, R.J. Mart, A.M. Smith, J.E. Gough, Bioresponsive hydrogels, Materials Today, 10 (2007) 40-48. LORENA GARCÍA DEL RÍO 56 [194] A. Hendi, M. Umair Hassan, M. Elsherif, B. Alqattan, S. Park, A.K. Yetisen, H. Butt, Healthcare Applications of pH-Sensitive Hydrogel-Based Devices: A Review, Int J Nanomedicine, 15 (2020) 3887-3901. [195] M. Rizwan, R. Yahya, A. Hassan, M. Yar, A.D. Azzahari, V. Selvanathan, F. Sonsudin, C.N. Abouloula, pH Sensitive Hydrogels in Drug Delivery: Brief History, Properties, Swelling, and Release Mechanism, Material Selection and Applications, Polymers (Basel), 9 (2017). [196] S. Bazban-Shotorbani, M.M. Hasani-Sadrabadi, A. Karkhaneh, V. Serpooshan, K.I. Jacob, A. Moshaverinia, M. Mahmoudi, Revisiting structure-property relationship of pH-responsive polymers for drug delivery applications, J Control Release, 253 (2017) 46-63. [197] S. Das, U. Subuddhi, pH-Responsive guar gum hydrogels for controlled delivery of dexamethasone to the intestine, Int J Biol Macromol, 79 (2015) 856-863. [198] D.S. Seeli, M. Prabaharan, Guar gum oleate-graftpoly(methacrylic acid) hydrogel as a colon-specific controlled drug delivery carrier, Carbohydr Polym, 158 (2017) 51-57. [199] Q. Wu, L. Wang, H. Yu, J. Wang, Z. Chen, Organization of glucose-responsive systems and their properties, Chem Rev, 111 (2011) 7855-7875. [200] D. Eleftheriadou, D. Kesidou, F. Moura, E. Felli, W. Song, Redox-Responsive Nanobiomaterials-Based Therapeutics for Neurodegenerative Diseases, Small, 16 (2020) e1907308. [201] H. Ye, Y. Zhou, X. Liu, Y. Chen, S. Duan, R. Zhu, Y. Liu, L. Yin, Recent Advances on Reactive Oxygen Species-Responsive Delivery and Diagnosis System, Biomacromolecules, 20 (2019) 24412463. [202] G. Saravanakumar, J. Kim, W.J. Kim, Reactive-OxygenSpecies-Responsive Drug Delivery Systems: Promises and Challenges, Adv Sci (Weinh), 4 (2017) 1600124. [203] Q. Xu, C. He, K. Ren, C. Xiao, X. Chen, Thermosensitive Polypeptide Hydrogels as a Platform for ROS-Triggered Cargo Release with Innate Cytoprotective Ability under Oxidative Stress, Adv Healthc Mater, 5 (2016) 1979-1990. 1.Introduction 57 [204] H. Zhao, J. Huang, Y. Li, X. Lv, H. Zhou, H. Wang, Y. Xu, C. Wang, J. Wang, Z. Liu, ROS-scavenging hydrogel to promote healing of bacteria infected diabetic wounds, Biomaterials, 258 (2020) 120286. [205] R. Zhang, A. Bowyer, R. Eisenthal, J. Hubble, A smart membrane based on an antigen-responsive hydrogel, Biotechnol Bioeng, 97 (2007) 976-984. [206] T. Miyata, T. Uragami, K. Nakamae, Biomolecule-sensitive hydrogels, Adv Drug Deliv Rev, 54 (2002) 79-98. [207] S.M. George, S. Tandon, B. Kandasubramanian, Advancements in Hydrogel-Functionalized Immunosensing Platforms, ACS Omega, 5 (2020) 2060-2068. [208] R. Chandrawati, Enzyme-responsive polymer hydrogels for therapeutic delivery, Exp Biol Med (Maywood), 241 (2016) 972-979. [209] J. Hu, G. Zhang, S. Liu, Enzyme-responsive polymeric assemblies, nanoparticles and hydrogels, Chem Soc Rev, 41 (2012) 5933-5949. [210] M. Zelzer, S.J. Todd, A.R. Hirst, T.O. McDonald, R.V. Ulijn, Enzyme responsive materials: design strategies and future developments, Biomater Sci, 1 (2013) 11-39. [211] M. Shahriari, M. Zahiri, K. Abnous, S.M. Taghdisi, M. Ramezani, M. Alibolandi, Enzyme responsive drug delivery systems in cancer treatment, J Control Release, 308 (2019) 172-189. [212] E. Verheyen, J.P. Schillemans, M. van Wijk, M.-A. Demeniex, W.E. Hennink, C.F. van Nostrum, Challenges for the effective molecular imprinting of proteins, Biomaterials, 32 (2011) 3008-3020. [213] M.E. Byrne, K. Park, N.A. Peppas, Molecular imprinting within hydrogels, Advanced Drug Delivery Reviews, 54 (2002) 149-161. [214] K. Edsman, H. Hagerstrom, Pharmaceutical applications of mucoadhesion for the non-oral routes, J Pharm Pharmacol, 57 (2005) 3-22. [215] A. Partenhauser, A. Bernkop-Schnürch, Mucoadhesive polymers in the treatment of dry X syndrome, Drug Discovery Today, 21 (2016) 1051-1062. LORENA GARCÍA DEL RÍO 64 - Assess the suitability, performance, and accuracy of artificial intelligence tools such as neurofuzzy logic to select suitable polymers and to establish the influence of each ingredient on the texturometric, rheological, and drug-release properties of the hydrogels. Results related to this objective are displayed in Chapter I: “Design of novel orotransmucosal vaccine-delivery platforms using artificial intelligence” and have already been published [1]. - Evaluate the utility of a hybrid AI software to optimize a sublingual hydrogel formulation presenting the desired texturometric, rheological and release properties. Furthermore, assess the utility of the optimized system as a Chlamydia vaccine carrier to elicit systemic and mucosal immune responses in mice. Results related to this objective are displayed in Chapter II: “Sublingual boosting with a novel mucoadhesive thermogelling hydrogel following parenteral CAF01 priming as a strategy against Chlamydia trachomatis”. Objective II: Design of an optimized, thermosensitive and highly mucoadhesive enema useful as a carrier of human cervical stem cells conditioned medium for the treatment of inflammatory bowel disease (IBD). - Demonstrate the utility of artificial intelligence tools to establish the design space of ternary hydrogel formulations with appropriate characteristics (thermosensitivity, mucoadhesivity, extensibility and retention in the rectal tract) for the treatment of IBD. Results related to this objective are displayed in Chapter III: “New tools to design smart thermosensitive hydrogels for protein rectal delivery in IBD” and have already been published [2]. - Evaluate the in vivo performance of the optimized hydrogel, loaded with conditioned medium from human uterine cervical stem cells in an experimental mouse model of acute colitis. Results related to this objective will be displayed in Chapter IV: “Tailored hydrogels as delivery platforms for conditioned medium from mesenchymal stem cells in a mouse model of acute colitis”. 2. Objetives 65 REFERENCES [1] L. Garcia-del Rio, P. Diaz-Rodriguez, M. Landin, Design of novel orotransmucosal vaccine-delivery platforms using artificial intelligence, European Journal of Pharmaceutics and Biopharmaceutics, 159 (2021) 3643. [2] L. Garcia-Del Rio, P. Diaz-Rodriguez, M. Landin, New tools to design smart thermosensitive hydrogels for protein rectal delivery in IBD, Mater Sci Eng C Mater Biol Appl, 106 (2020) 110252. 3. CHAPTER I: DESIGN OF NOVEL OTROTRANSMUCOSAL VACCINE DELIVERY PLATFORMS USING ARTIFICIAL INTELLIGENCE *The results from this chapter and its corresponding annex, have already been published as L. Garcia-del Rio1, P. Diaz-Rodriguez2, M. Landin1, Design of novel orotransmucosal vaccine-delivery platforms using artificial intelligence, European Journal of Pharmaceutics and Biopharmaceutics, 159 (2021) 36-43. ISSN: 0939-6411; E-ISSN:1873-0191. 1R+D Pharma Group (GI-1645), Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Facultad de Farmacia, Universidade de Santiago de Compostela-Campus Vida, 15782, Santiago de Compostela, Spain. 2Departament of Chemical Engineering and Pharmaceutical Technology, Facultad de Ciencias de la Salud, Universidad de La Laguna (ULL), Campus de Anchieta, 38200, La Laguna, Spain. Chapter I: Design of novel orotransmucosal vaccine delivery platforms using artificial intelligence 69 ABSTRACT The linings of the oral cavity are excellent needle-free vaccination sites, able to induce immune responses at distal sites and confer systemic protection. However, owing to the mucosal tissues’ intrinsic characteristics, the design of effective antigen-delivery systems is not an easy task. In the present work, we propose to develop and characterize thermosensitive and mucoadhesive hydrogels for orotransmucosal vaccination taking advantage of artificial intelligence tools (AIT). Hydrogels of variable composition were obtained combining Pluronic® F127 (PF127), Hybrane® S1200 (HS1200) and Gantrez® AN119 (AN119) or S97 (S97). Systems were characterized in terms of physicochemical properties, adhesion capacity to mucosal tissues and antigen-like microspheres release. Additionally, polymers biocompatibility and their immune-stimulation capacity were assessed in human macrophages. Interestingly, cells treated with HS1200 exhibited a significant proliferation enhancement compared to control. The use of AIT allowed to determine the effect of each polymer on formulations properties. The used proportions of PF127 and Gantrez® are mainly the factors controlling gelation temperature, mucoadhesion, adhesion work and gel strength. Meanwhile, cohesion and short-term microsphere release are dependent on the PF127 concentration. However, long-term microsphere release varies depending on the Gantrez® variety and the PF127 concentration used. Hydrogels prepared with S97 showed slower microsphere release. The use of AIT allowed to establish the conditions able to produce ternary hydrogels with immunostimulatory properties together with adequate mucoadhesion capacity and antigen-like microspheres release. Chapter I: Design of novel orotransmucosal vaccine delivery platforms using artificial intelligence 71 3.1. INTRODUCTION The rise of biologics has intensified the interest on employing the mucosal surfaces as administration sites. As an example, peptides have been already successfully delivered through the nasal route. However, this administration approach presents more limitations than the oral cavity mucous membranes such as lower absorption surface and higher susceptibility to irreversible tissue damage or irritation [1, 2]. Therefore, the linings of the oral cavity have been suggested as excellent alternative sites for needleless administration of biomacromolecules [2-7]. Currently, sublingual and buccal routes are the most commonly used for non-invasive orotransmucosal delivery of biomacromolecules [8]. On these administration routes the drug absorption takes place through non-keratinized tissues characterized by low enzymatic activity, good vascularization that improves systemic bioavailability and relatively high permeability. Moreover, the nature of the oral cavity allows easy access for administration and removal in case of adverse events. Furthermore, both epithelia can induce immune responses at several distal sites, being suitable for vaccine administration [1, 2, 9-12]. Thus, different dosage forms such as sprays, films, droplets, tablets and hydrogels have been designed as vaccine carriers for orotransmucosal delivery [12-14]. Compared to parenteral vaccination, mucosal immunization is able to elicit stronger and more effective immune responses at mucosal sites together with systemic immunity. Moreover, it does not require specialized staff for administration and allows greater patient compliance [15, 16]. However, the development of suitable mucosal vaccines is extremely challenging, due to the mucosal tissues’ intrinsic characteristics [16, 17]. Besides, the characteristics of the selected immunostimulatory molecules should be taken into account when designing new vaccine delivery platforms. For infectious diseases’ vaccines these molecules can be specific antigens or the whole organism [18]. Whole inactivated or attenuated bacteria stimulate the immune system by promoting their phagocytosis and presentation by APCs (antigen-presenting cells). From the technological point of view, the bacteria suspensions used in vaccine formulations behave like a microparticulated system. LORENA GARCÍA DEL RÍO 72 Among the suitable vaccine carriers for orotransmucosal delivery, hydrogels stand out by their capacity to allow homogenous particle dispersion. Moreover, hydrogels present several advantages as ability to be distributed over a wide area of the oral cavity minimizing the risk of drug low retention and delivery deficiency [11, 19]. More specifically, thermosensitive mucoadhesive hydrogels can form hydrogen bonds with mucins, dramatically increasing the residence time of the formulation in contact with the mucosa [20]. Furthermore, their thermogelling character ensure their easy administration while reducing their washing by saliva. These benefits make mucoadhesive and thermosensitive hydrogels good and feasible candidates for orotransmucosal antigen delivery. Hydrogels can be obtained combining diverse polymers, which depending on their nature and concentration, will give to systems with variable characteristics. Pluronic® are amphiphilic copolymers capable of self-assembly, generating physical crosslinked hydrogels in aqueous media above their critical micellar concentration (CMC) when they reach a certain temperature [20]. PF127 variety has been widely used in the vaccine delivery field, especially for subcutaneous formulations due to its well-known sol-gel transition at body temperature, which allows an easy handling together with a depot effect at the injection site [21-23]. Thereby, PF127 would confer thermosensitive properties to hydrogels, obtaining liquid systems ease to apply in the oral cavity that become solid at body temperature. Furthermore, its immunomodulatory properties have been previous reported [24]. Hyperbranched polymers are characterized by their tree-like globular topology and large number of functional groups, which confers them high reactivity. The high crosslinking densities of these polymers constitutes its main advantage, leading to well integrated polymeric networks with low viscosity [25]. Hybrane® S1200 is a hyperbranched polyesteramide with various multifunctional end groups (e.g. hydroxyl groups) conferring potential mucoadhesive properties [26]. On the other hand, Gantrez® or poly(methyl vinyl ether-co-maleic) anhydride (AN) or acid (S) are well-known high bioadhesive anionic amphiphilic copolymers [27, 28]. These materials Chapter I: Design of novel orotransmucosal vaccine delivery platforms using artificial intelligence 73 are widely employed in the pharmaceutical field as suspending and thickening agents, denture adhesives and adjuvants for the preparation of transdermal patches. They have been previously used in the field of vaccine delivery [29-32]. In this work we propose to combine Pluronic® F127, Hybrane® S1200 and Gantrez® S97 or AN119 to obtain ternary thermosensitive mucoadhesive hydrogels. The design of complex multicomponent systems involves many difficulties associated with the number of variables implicated. Recently, Artificial Neural Networks (ANN) combined with other Artificial Intelligence Techniques (AIT) as genetic algorithms and fuzzy logic have been proposed as useful approaches to develop smart hydrogels with desired properties using extremely reduced experimental designs [33]. AIT modelling allow to understand how final pharmaceutical system properties are affected by the ingredients and operation conditions [34-36]. Thereby, the aim of the present work is to develop suitable thermosensitive and mucoadhesive hydrogels for orotransmucosal delivery of whole attenuated microbes. To achieve this goal, the workflow can be summarized as follows: a) test the potential in vitro immunostimulatory capacity of the polymers selected; b) evaluate the suitability of ternary compositions to achieve hydrogels with appropriate thermosensitive and mucoadhesive properties; c) compare the properties of the hydrogels elaborated with Gantrez® AN119 and S97; and d) select the hydrogel composition able to meet all the requirements for orotransmucosal vaccine-delivery. AIT were employed to model the properties of ternary systems, understand their behaviour and explore their design space. 3.2. MATERIALS AND METHODS 3.2.1. Materials Pluronic® F127 (PF127) was obtained from Sigma-Aldrich (USA). Hybrane® S1200 (HS1200) was purchased from Polymer Factory (Sweden). Gantrez® AN119 (AN119) and Gantrez® S97 (S97) were kindly provided by Ashland (Spain). Fluoro-MaxTM Green fluorescent microspheres (1 µm) were acquired from Thermo Scientific (USA). Cell Strainers presenting a pore size of 40 µm were LORENA GARCÍA DEL RÍO 176 Figure 8. Representative histological score in the distal colon fragment (continuation). Microphotography of representative examples of inflammation, atrophy, and dysplasia in DSS-induced mice non-treated (NT) and treated with H-DMEM-F12, H-hUCESC-CM, and mesalazine (MZ). *=p<0.05; ns=not significant. Scale bar: 100 mm. 6.3.5. H-hUCESC-CM pro-inflammatory cytokines in colon TNF-α, IL-6, and IFN-γ mRNA expression levels in the different mouse groups are shown in Figure 9. TNF-α, IFN-γ and IL-6 mRNA expression levels were significantly higher in the NT mice with respect to H mice (p<0.0001, p<0.01, and p<0.0001, respectively). All the cytokines evaluated significantly decreased after treatment with HhUCESC-CM as compared with NT mice (p<0.0001). Importantly, TNF-α, IFN-γ and IL-6 mRNA expression levels of H mice were similar (not significant) to both H-hUCESC-CM and MZ treated mice. 6.Chapter IV: Tailored hydrogels as delivery platforms for conditioned medium from mesenchymal stem cells in a mouse model of acute colitis 177 Figure 9. Real-time PCR of pro-inflammatory cytokines in colon samples. A) Tumour necrosis factor-alpha (TNF-α), B) Interferon-gamma (IFN-γ) and C) Interleukin-6 (IL-6) mRNA expression levels in colon of mice. Data are expressed as mean ± SEM. ns= not significant. **=p<0.01; ***=p<0.001. 6.4. DISCUSSION In the present study we demonstrate that local administration of HhUCESC-CM has therapeutic effects in an acute DSS-induced colitis mouse model. These beneficial effects comprise body weight recovery, improved colon length and histological colitis score, and decreased pro-inflammatory cytokine expression. LORENA GARCÍA DEL RÍO 178 Mucosal membranes are excellent sites for drug delivery owing to their high permeability, lower risk of overdose and local treatment. However, the major drawback of drug delivery via mucosal membranes is the limited retention time at the mucosal tissue surface [25,26]. To address this limitation an adequate ternary formulation for rectal administration was developed to evaluate the potential usefulness of hUCESC-CM for IBD treatment. This system is composed of two Pluronic® and a hydroxypropylcellulose (MK4M). Pluronic® are amphiphilic, thermosensitive and bioadhesive triblock copolymers widely used in the pharmaceutical field to produce in situ gelation hydrogels [27]. Often, PF127 and PF68 are used in combination to obtain hydrogels with specific gelation temperatures [28]. Hydroxypropylcellulose is a cellulose derivative with high viscosity grade that generates robust and mucoadhesive networks capable of controlling the release of drugs. Thus, the combination of these compounds should increase the binding of the hydrogel to the colonic and rectal mucosae [29]. The use of ANN made it possible to obtain an optimized bioadhesive hydrogel that was stable and suitable for rectal administration of hUCESC-CM. Its mucoadhesive and thermosensitive character together with its suitable syringeability ensures adequate administration and extensibility in liquid form on the colonic tissue. The low Tgel (27 °C) of H-DMEM-F12 enables a faster transition from liquid to solid state by the Pluronic® polymeric chains rearrangement, reducing leakage and dilution of the systems in biological fluids [27]. Moreover, the incorporation of MK4M improves hydrogel bioadhesion for a longer and closer interaction between hUCESC-CM and colonic and rectal mucosae [27,30]. Thus, the residence time of the drug delivery system in the colon increases, enhancing drug absorption, as well as efficacy and bioavailability. With respect to NT mice, we found that H-hUCESC-CM treatment significantly improved several histopathological parameters, such as colon length and histological score. Specifically, the lower grade of atrophy, inflammation and dysplasia suggests that HhUCESC-CM acts upon several targets. It is known that molecular and cellular mechanisms responsible for MSC-mediated attenuation of 6.Chapter IV: Tailored hydrogels as delivery platforms for conditioned medium from mesenchymal stem cells in a mouse model of acute colitis 179 murine colitis involved suppression of colon inflammation, promotion of angiogenesis and regeneration of damaged epithelium, leading to an enhanced healing process in the injured colon [31-33]. It is worth noting that, compared to the standard formulation (MZ), treatment with H-hUCESC-CM improved histological score only at distal colon, which is interesting because histopathological damage by DSS is predominantly found in distal colonic location [34]. It has been previously demonstrated in a dry eye rat model that hUCESC-CM treatment significantly improves epithelial regeneration and decreases pro-inflammatory cytokines [18]. hUCESC-CM contains high levels of proteins such as tissue inhibitors of metalloproteinases -1 and -2, fibroblast growth factor -6 and -7, urokinase receptor, and hepatocyte growth factor, that could mediate regenerative effects [17-20]. In the present study, it was also found that mice treated with H-hUCESC-CM showed a significant decrease in TNF-α, IL-6 and INF-γ mRNA expression. TNF-α is considered the most potent pro-inflammatory cytokine in the pathogenesis of IBD. In fact, anti-TNF-α agents, such as infliximab, are highly effective in the treatment of moderate to severe intestinal disease [35]. Experimental data show that hydrogel alone also conferred a partial benefit on some pro-inflammatory cytokines such as TNF-α and IFN-γ. Multiple biological effects are attributed to PF68 and to PF127, such as membrane resealing agent, antithrombotic, skin wound cleaner, and emulsifying agent in artificial blood [36,37]. Hence, partial benefit observed in colon of mice treated with H-DMEM-F12 could be explained by partial anti-inflammatory and resealing properties of the polymers included in the formulation. Earlier studies attributed the effects of MSC therapies to their capacity for local engrafting and differentiating into multiple tissue types, however, more recent studies show that implanted cells do not survive for long [38,39]. This suggests that the beneficial effects of MSC therapy could be derived from secreted bioactive factors regulating key biologic processes [40]. Therefore, secretome derivatives such as CM or exosomes, may be preferable to cell administration in terms of manufacture, storage and handling. The use of MSC-CM mitigates certain safety considerations related to the LORENA GARCÍA DEL RÍO 180 transplantation of living cells such as immune compatibility, emboli formation, tumorigenicity, and transmission of infections [41]. One limitation of this study is that only a single concentration of HhUCESC-CM was evaluated. It is known that the effectiveness of this type of therapy may vary depending on the dose and frequency of administration. Further research is necessary to elucidate these aspects as well as underlying mechanisms and components of hUCESC-CM. 6.5. CONCLUSIONS This study demonstrates that local administration of hUCESC-CM in bioadhesive thermosensitive hydrogels improves residence time in target sites and increases interaction with rectal and colonic mucosae, inducing therapeutic anti-inflammatory and regenerative activity in experimental colitis. Thus, these data suggest that H-hUCESC-CM may represent an attractive tool for cell-free therapy in IBD local treatment. 6.Chapter IV: Tailored hydrogels as delivery platforms for conditioned medium from mesenchymal stem cells in a mouse model of acute colitis 181 REFERENCES [1] J. Cosnes, C. Gower-Rousseau, P. Seksik, A. Cortot, Epidemiology and natural history of inflammatory bowel diseases, Gastroenterology, 140 (2011) 1785-1794. [2] J. Burisch, N. Pedersen, S. Cukovic-Cavka, M. Brinar, I. Kaimakliotis, D. Duricova, O. Shonova, I. Vind, S. Avnstrom, N. Thorsgaard, V. Andersen, S. Krabbe, J.F. Dahlerup, R. Salupere, K.R. Nielsen, J. Olsen, P. Manninen, P. Collin, E.V. Tsianos, K.H. Katsanos, K. Ladefoged, L. Lakatos, E. Bjornsson, G. Ragnarsson, Y. Bailey, S. Odes, D. Schwartz, M. Martinato, G. Lupinacci, M. Milla, A. De Padova, R. D'Inca, M. Beltrami, L. Kupcinskas, G. Kiudelis, S. Turcan, O. Tighineanu, I. Mihu, F. Magro, L.F. Barros, A. Goldis, D. Lazar, E. Belousova, I. Nikulina, V. Hernandez, D. Martinez-Ares, S. Almer, Y. Zhulina, J. Halfvarson, N. Arebi, S. Sebastian, P.L. Lakatos, E. Langholz, P. Munkholm, g. EpiCom, East-West gradient in the incidence of inflammatory bowel disease in Europe: the ECCOEpiCom inception cohort, Gut, 63 (2014) 588-597. [3] G.J. Gomez-Gomez, A. Masedo, C. Yela, P. Martinez-Montiel Mdel, B. Casis, Current stage in inflammatory bowel disease: What is next?, World J Gastroenterol, 21 (2015) 11282-11303. [4] B. Chassaing, A. Darfeuille-Michaud, The commensal microbiota and enteropathogens in the pathogenesis of inflammatory bowel diseases, Gastroenterology, 140 (2011) 1720-1728. [5] T.T. MacDonald, I. Monteleone, M.C. Fantini, G. Monteleone, Regulation of homeostasis and inflammation in the intestine, Gastroenterology, 140 (2011) 1768-1775. [6] A. Thomas, N. Lodhia, Advanced therapy for inflammatory bowel disease: a guide for the primary care physician, J Am Board Fam Med, 27 (2014) 411-420. [7] S. Mozaffari, S. Nikfar, A.H. Abdolghaffari, M. Abdollahi, New biologic therapeutics for ulcerative colitis and Crohn's disease, Expert Opin Biol Ther, 14 (2014) 583-600. [8] H.E. Young, M.L. Mancini, R.P. Wright, J.C. Smith, A.C. Black, Jr., C.R. Reagan, P.A. Lucas, Mesenchymal stem cells reside within the connective tissues of many organs, Dev Dyn, 202 (1995) 137-144. LORENA GARCÍA DEL RÍO 182 [9] S. Meirelles Lda, A.M. Fontes, D.T. Covas, A.I. Caplan, Mechanisms involved in the therapeutic properties of mesenchymal stem cells, Cytokine Growth Factor Rev, 20 (2009) 419-427. [10] S.K. Hosseini-Asl, D. Mehrabani, F.K. Karimi-Busheri, Therapeutic effect of mesenchymal stem cells in ulcerative colitis: a review on achievements and challenges. J Clin Med, 9 (2020) 3922. [11] X. Shi, Q. Chen, F. Wang, Mesenchymal stem cells for the treatment of ulcerative colitis: a systematic review and meta-analysis of experimental and clinical studies, Stem Cell Res Ther, 10 (2019) 266. [12] E. Legaki, M.G. Roubelakis, G.E. Theodoropoulos, A. Lazaris, A. Kollia, G. Karamanolis, E. Marinos, M. Gazouli, Therapeutic Potential of Secreted Molecules Derived from Human Amniotic Fluid Mesenchymal Stem/Stroma Cells in a Mice Model of Colitis, Stem Cell Rev, 12 (2016) 604-612 [13] J.Y. Song, H.J. Kang, J.S. Hong, C.J. Kim, J.Y. Shim, C.W. Lee, J. Choi, Umbilical cord-derived mesenchymal stem cell extracts reduce colitis in mice by re-polarizing intestinal macrophages, Sci Rep, 7 (2017) 9412. [14] Y. Wu, W. Qiu, X. Xu, J. Kang, J. Wang, Y. Wen, X. Tang, Y. Yan, H. Qian, X. Zhang, W. Xu, F. Mao, Exosomes derived from human umbilical cord mesenchymal stem cells alleviate inflammatory bowel disease in mice through ubiquitination, Am J Transl Res, 10 (2018) 2026-2036 [15] J. Panes, D. Garcia-Olmo, G. Van Assche, J.F. Colombel, W. Reinisch, D.C. Baumgart, A. Dignass, M. Nachury, M. Ferrante, L. Kazemi-Shirazi, J.C. Grimaud, F. de la Portilla, E. Goldin, M.P. Richard, A. Leselbaum, S. Danese, A.C.S.G. Collaborators, Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn's disease: a phase 3 randomised, double-blind controlled trial, Lancet, 388 (2016) 1281-1290. [16] J. Panes, D. Garcia-Olmo, G. Van Assche, J.F. Colombel, W. Reinisch, D.C. Baumgart, A. Dignass, M. Nachury, M. Ferrante, L. Kazemi-Shirazi, J.C. Grimaud, F. de la Portilla, E. Goldin, M.P. Richard, M.C. Diez, I. Tagarro, A. Leselbaum, S. Danese, A.C.S.G. Collaborators, Long-term Efficacy and Safety of Stem Cell Therapy 6.Chapter IV: Tailored hydrogels as delivery platforms for conditioned medium from mesenchymal stem cells in a mouse model of acute colitis 183 (Cx601) for Complex Perianal Fistulas in Patients With Crohn's Disease, Gastroenterology, 154 (2018) 1334-1342 e1334. [17] N. Eiro, J. Sendon-Lago, S. Seoane, M.A. Bermudez, M.L. Lamelas, T. Garcia-Caballero, J. Schneider, R. Perez-Fernandez, F.J. Vizoso, Potential therapeutic effect of the secretome from human uterine cervical stem cells against both cancer and stromal cells compared with adipose tissue stem cells, Oncotarget, 5 (2014) 1069210708. [18] M.A. Bermudez, J. Sendon-Lago, N. Eiro, M. Trevino, F. Gonzalez, E. Yebra-Pimentel, M.J. Giraldez, M. Macia, M.L. Lamelas, J. Saa, F. Vizoso, R. Perez-Fernandez, Corneal epithelial wound healing and bactericidal effect of conditioned medium from human uterine cervical stem cells, Invest Ophthalmol Vis Sci, 56 (2015) 983-992. [19] M.A. Bermudez, J. Sendon-Lago, S. Seoane, N. Eiro, F. Gonzalez, J. Saa, F. Vizoso, R. Perez-Fernandez, Anti-inflammatory effect of conditioned medium from human uterine cervical stem cells in uveitis, Exp Eye Res, 149 (2016) 84-92. [20] J. Sendon-Lago, S. Seoane, A. Martinez-Ordonez, N. Eiro, J. Saa, F.J. Vizoso, F. Gonzalez, R. Perez-Fernandez, M.A. Bermudez, Corneal regeneration by conditioned medium of human uterine cervical stem cells is mediated by TIMP-1 and TIMP-2, Exp Eye Res, 180 (2019) 110-121. [21] J. Schneider, E. Mateo, C. Marcos-Arias, N. Eiro, F. Vizoso, R. Perez-Fernandez, E. Eraso, G. Quindos, Antifungal Activity of the Human Uterine Cervical Stem Cells Conditioned Medium (hUCESCCM) Against Candida albicans and Other Medically Relevant Species of Candida, Front Microbiol, 9 (2018) 2818. [22] L. Garcia-Del Rio, P. Diaz-Rodriguez, M. Landin, New tools to design smart thermosensitive hydrogels for protein rectal delivery in IBD, Mater Sci Eng C Mater Biol Appl, 106 (2020) 110252. [23] S. Wirtz, V. Popp, M. Kindermann, K. Gerlach, B. Weigmann, S. Fichtner-Feigl, M.F. Neurath, Chemically induced mouse models of acute and chronic intestinal inflammation, Nat Protoc, 12 (2017) 1295-1309. LORENA GARCÍA DEL RÍO 184 [24] H.S. Cooper, S.N. Murthy, R.S. Shah, D.J. Sedergran, Clinicopathologic study of dextran sulfate sodium experimental murine colitis, Lab Invest, 69 (1993) 238-249. [25] H. Laroui, S.A. Ingersoll, H.C. Liu, M.T. Baker, S. Ayyadurai, M.A. Charania, F. Laroui, Y. Yan, S.V. Sitaraman, D. Merlin, Dextran sodium sulfate (DSS) induces colitis in mice by forming nanolipocomplexes with medium-chain-length fatty acids in the colon, PLoS One, 7 (2012) e32084. [26] A.A. Koffi, F. Agnely, G. Ponchel, J.L. Grossiord, Modulation of the rheological and mucoadhesive properties of thermosensitive poloxamer-based hydrogels intended for the rectal administration of quinine, Eur J Pharm Sci, 27 (2006) 328-335. [27] Y. Yuan, Y. Cui, L. Zhang, H.P. Zhu, Y.S. Guo, B. Zhong, X. Hu, L. Zhang, X.H. Wang, L. Chen, Thermosensitive and mucoadhesive in situ gel based on poloxamer as new carrier for rectal administration of nimesulide, Int J Pharm, 430 (2012) 114-119. [28] F.U. Din, O. Mustapha, D.W. Kim, R. Rashid, J.H. Park, J.Y. Choi, S.K. Ku, C.S. Yong, J.O. Kim, H.G. Choi, Novel dual-reverse thermosensitive solid lipid nanoparticle-loaded hydrogel for rectal administration of flurbiprofen with improved bioavailability and reduced initial burst effect, Eur J Pharm Biopharm, 94 (2015) 64-72. [29] A.C. Santos Akkari, E.V. Ramos Campos, A.F. Keppler, L.F. Fraceto, E. de Paula, G.R. Tofoli, D.R. de Araujo, Budesonidehydroxypropyl-beta-cyclodextrin inclusion complex in binary poloxamer 407/403 system for ulcerative colitis treatment: A physicochemical study from micelles to hydrogels, Colloids Surf B Biointerfaces, 138 (2016) 138-147. [30] N. Salamat-Miller, M. Chittchang, T.P. Johnston, The use of mucoadhesive polymers in buccal drug delivery, Adv Drug Deliv Rev, 57 (11) 1666-1691. [31] J. Yang, Z. Chen, D. Pan, H. Li, J. Shen, Umbilical Cord-Derived Mesenchymal Stem Cell-Derived Exosomes Combined Pluronic F127 Hydrogel Promote Chronic Diabetic Wound Healing and Complete Skin Regeneration, Int J Nanomedicine, 15 (2020) 5911-5926. 6.Chapter IV: Tailored hydrogels as delivery platforms for conditioned medium from mesenchymal stem cells in a mouse model of acute colitis 185 [32] P. Ahangar, S. J. Mills, A.J. Cowin, Mesenchymal Stem Cell Secretome as an Emerging Cell-Free Alternative for Improving Wound Repair, Int J Mol Sci, 21 (2020) 7038. [33] Q.Q. Chen, L. Yan, C.Z. Wang, W.H. Wang, H. Shi, B.B. Su, Q.H. Zeng, H.T. Du, J. Wan, Mesenchymal stem cells alleviate TNBS-induced colitis by modulating inflammatory and autoimmune responses, World J Gastroenterol, 19 (2013) 4702-4717. [34] L.A. Dieleman, M.J. Palmen, H, Akol, E. Bloemena, A.S. Peña, S.G. Meuwissen, E.P. Van Rees, Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines, Clin Exp Immunol 114 (1998) 385-391. [35] S. Danese, C. Fiocchi, Ulcerative colitis, N Engl J Med, 365 (2011) 1713-1725. [36] G.M. Soliman, G. Fetih, A.M. Abbas, Thermosensitive bioadhesive gels for the vaginal delivery of sildenafil citrate: in vitro characterization and clinical evaluation in women using clomiphene citrate for induction of ovulation, Drug Dev Ind Pharm, 43 (2017) 399-408. [37] Y. Liu, W-L. Lu, J-C. Wang, X. Zhang, H. Zhang, X-Q. Wang, T-Y. Zhou, Q. Zhang, Controlled delivery of recombinant hirudin based on thermo-sensitive Pluronic F127 hydrogel for subcutaneous administration: In vitro and in vivo characterization, J Control Release, 117 (2007) 387-95. [38] G. Maguire, Stem cell therapy without the cells, Commun Integr Biol, 6 (2013) e26631. [39] C.F. de Aguiar, A. Castoldi, V. Andrade-Oliveira, A. Ignacio, F.F. da Cunha, R.J.F. Felizardo, E.J. Bassi, N.O.S. Camara, D.C. de Almeida, Mesenchymal stromal cells modulate gut inflammation in experimental colitis, Inflammopharmacology, 26 (2018) 251-260. [40] F.J. Vizoso, N. Eiro, L. Costa, P. Esparza, M. Landin, P. DiazRodriguez, J. Schneider, R. Perez-Fernandez, Mesenchymal stem cells in homeostasis and systemic diseases: hypothesis, evidences, and therapeutic opportunities, Int J Mol Sci, 20 (2019) 3738. [41] F.J. Vizoso, N. Eiro, S. Cid, J. Schneider, R. Perez-Fernandez, Mesenchymal stem cell secretome: toward cell-free therapeutic ANNEX I Annex I 195 Table A1.1. Set of “IF-THEN” rules for gelation temperature for the experimental design obtained through AIT. Degree of membership in parentheses. Blue colour indicates the combination of inputs that led to the highest value of the output, while red colour shows the combination of inputs giving the lowest value. Table A1.2. Set of “IF-THEN” rules for mucoadhesion at 37 ⁰C for the experimental design obtained through AIT. Degree of membership in parentheses. Blue colour indicates the combination of inputs that led to the highest value of the output, while red colour shows the combination of inputs giving the lowest value. Rules for Tgel Submodel 1 IF PF127 is LOW AND Gantrez is LOW_1(4) THEN Tgel is LOW (0.65) IF PF127 is LOW AND Gantrez is MID_2(4) THEN Tgel is LOW (0.82) IF PF127 is LOW AND Gantrez is MID_3(4) THEN Tgel is HIGH (0.82) IF PF127 is LOW AND Gantrez is HIGH_4(4) THEN Tgel is HIGH (1.00) IF PF127 is MID AND Gantrez is LOW_1(4) THEN Tgel is HIGH (0.97) IF PF127 is MID AND Gantrez is MID_2(4 THEN Tgel is LOW (1.00) IF PF127 is MID AND Gantrez is MID_3(4) THEN Tgel is LOW (1.00) IF PF127 is MID AND Gantrez is HIGH_4(4) THEN Tgel is LOW (0.72) IF PF127 is HIGH AND Gantrez is LOW_1(4 THEN Tgel is LOW (1.00) IF PF127 is HIGH AND Gantrez is MID_2(4) THEN Tgel is LOW (1.00) IF PF127 is HIGH AND Gantrez is MID_3(4) THEN Tgel is LOW (1.00) IF PF127 is HIGH AND Gantrez is HIGH_4(4) THEN Tgel is LOW (1.00) Submodel 2 IF HS1200 is LOW THEN Tgel is LOW (0.56) IF HS1200 is HIGH THEN Tgel is LOW (0.51) Rules for Mucoadhesion Submodel 1 IF PF127 is LOW_1(4) AND Gantrez is LOW THEN Mucoadhesion is LOW(1.00) IF PF127 is LOW_1(4) AND Gantrez is HIGH THEN Mucoadhesion is LOW (0.93) IF PF127 is MID_2(4) AND Gantrez is LOW THEN Mucoadhesion is HIGH (0.70) IF PF127 is MID_2(4) AND Gantrez is HIGH THEN Mucoadhesion is LOW (0.87) IF PF127 is MID_3(4) AND Gantrez is LOW THEN Mucoadhesion is HIGH (1.00) IF PF127 is MID_3(4) AND Gantrez is HIGH THEN Mucoadhesion is HIGH (1.00) IF PF127 is HIGH_4(4) AND Gantrez is LOW THEN Mucoadhesion is HIGH (0.70) IF PF127 is HIGH_4(4) AND Gantrez is HIGH THEN Mucoadhesion is HIGH (0.82) LORENA GARCÍA DEL RÍO 196 Table A1.3. Set of “IF-THEN” rules for gel strength at 37 ⁰C for the experimental design obtained through AIT. Degree of membership in parentheses. Blue colour indicates the combination of inputs that led to the highest value of the output, while red colour shows the combination of inputs giving the lowest value. Table A1.4. Set of “IF-THEN” rules for cohesion at 37 ⁰C for the experimental design obtained through AIT. Degree of membership in parentheses. Blue colour indicates the combination of inputs that led to the highest value of the output, while red colour shows the combination of inputs giving the lowest value Rules for Gel strength Submodel 1 IF PF127 is LOW AND Gantrez is LOW_1(4) THEN Gel strength is HIGH (1.00) IF PF127 is LOW AND Gantrez is MID_2(4) THEN Gel strength is HIGH (0.86) IF PF127 is LOW AND Gantrez is MID_3(4) THEN Gel strength is LOW (1.00) IF PF127 is LOW AND Gantrez is HIGH_4(4) THEN Gel strength is LOW (1.00) IF PF127 is MID AND Gantrez is LOW_1(4) THEN Gel strength is LOW (1.00) IF PF127 is MID AND Gantrez is MID_2(4 THEN Gel strength is LOW (1.00) IF PF127 is MID AND Gantrez is MID_3(4) THEN Gel strength is LOW (1.00 IF PF127 is MID AND Gantrez is HIGH_4(4) THEN Gel strength is LOW (1.00 IF PF127 is HIGH AND Gantrez is LOW_1(4) THEN Gel strength is HIGH (1.00) IF PF127 is HIGH AND Gantrez is MID_2(4) THEN Gel strength is HIGH (1.00) IF PF127 is HIGH AND Gantrez is MID_3(4) THEN Gel strength is LOW (1.00 IF PF127 is HIGH AND Gantrez is HIGH_4(4) THEN Gel strength is HIGH (1.00) Submodel 2 IF HS1200 is LOW THEN Gel strength is HIGH (0.58) IF HS1200 is HIGH THEN Gel strength is LOW (0.78) Submodel 3 IF Gantrez type is S97 THEN Gel strength is HIGH (0.50) IF Gantrez type is AN119 THEN gel strength is LOW (0.70) Rules for Cohesion Submodel 1 IF PF127 is LOW THEN Cohesion is HIGH (1.00) IF PF127 is HIGH THEN Cohesion is LOW (0.95) Annex I 197 Table A1.5. Set of “IF-THEN” rules for adhesion work at 37 ⁰C for the experimental design obtained through AIT. Degree of membership in parentheses. Blue colour indicates the combination of inputs that led to the highest value of the output, while red colour shows the combination of inputs giving the lowest value. Table A1.6. Set of “IF-THEN” rules for GMFreleased at 5 minutes at 37 ⁰C for the experimental design obtained through AIT. Degree of membership in parentheses. Blue colour indicates the combination of inputs that led to the highest value of the output, while red colour shows the combination of inputs giving the lowest value Rules for Adhesion work Submodel 1 IF PF127 is LOW AND Gantrez is LOW THEN Adhesion work is LOW (0.85) IF PF127 is LOW AND Gantrez is MID THEN Adhesion work is LOW (0.77) IF PF127 is LOW AND Gantrez is HIGH THEN Adhesion work is LOW (1.00) IF PF127 is MID AND Gantrez is LOW THEN Adhesion work is LOW (1.00) IF PF127 is MID AND Gantrez is MID THEN Adhesion work is HIGH (0.65) IF PF127 is MID AND Gantrez is HIGH THEN Adhesion work is LOW (1.00) IF PF127 is HIGH AND Gantrez is LOW THEN Adhesion work is HIGH (1.00) IF PF127 is HIGH AND Gantrez is MID THEN Adhesion work is HIGH (1.00) IF PF127 is HIGH AND Gantrez is HIGH THEN Adhesion work is HIGH (1.00) Submodel 2 IF Gantrez type S97 THEN Adhesion work is LOW (0.57) IF Gantrez type AN119 THEN Adhesion work is LOW (0.84) Submodel 3 IF HS1200 is LOW THEN Adhesion work is LOW (0.59) IF HS1200 is HIGH THEN Adhesion work is LOW (0.82) Rules for GMF released at 5 minutes Submodel 1 IF PF127 is LOW THEN GMF released at 5 min is HIGH (0.83) IF PF127 is MID THEN GMF released at 5 min is HIGH (1.00) IF PF127 is HIGH THEN GMF released at 5 min is LOW (1.00) Submodel 2 IF Gantrez is LOW THEN GMF released at 5 min is HIGH (0.95) IF Gantrez is MID THEN GMF released at 5 min is LOW (0.75) IF Gantrez is HIGH THEN GMF released at 5 min is LOW (0.50) LORENA GARCÍA DEL RÍO 198 Table A1.7. Set of “IF-THEN” rules for total GMF release time at 37 ⁰C for the experimental design obtained through AIT. Degree of membership in parentheses. Blue colour indicates the combination of inputs that led to the highest value of the output, while red colour shows the combination of inputs giving the lowest value Rules for Total GMF release time Submodel 1 IF PF127 is LOW AND Gantrez type is S97 THEN Total release time is LOW (1.00) IF PF127 is LOW AND Gantrez type is AN119 THEN Total release time is HIGH (0.58) IF PF127 is MID AND Gantrez type S97 THEN Total release time is LOW (1.00) IF PF127 is MID AND Gantrez type is AN119 THEN Total release time is LOW (1.00) IF PF127 is HIGH AND Gantrez type is S97 THEN Total release time is HIGH (1.00) IF PF127 is HIGH AND Gantrez type is AN119 THEN Total release time is HIGH (0.74) Submodel 2 IF Gantrez is LOW THEN Total release time is LOW (1.00) IF Gantrez is MID THEN Total release time is LOW (0.61) IF Gantrez is HIGH THEN Total release time is HIGH (0.59) Submodel 3 IF HS1200 is LOW THEN Total release time is LOW (0.64) IF HS1200 is MID THEN Total release time is LOW (1.00) IF HS1200 is HIGH THEN Total release time is HIGH (0.54) Annex I 199 Figure A1. Effect of HS1200 treatments on cell proliferation of THP-1 derived macrophages after incubation for 24 h. Assays were performed in triplicate and results are expressed as mean ± SD. * denotes statistically significant differences (p < 0.05) compared to control, human macrophages without treatment. 0 20 40 60 80 100 120 140 160 0.00 0.25 0.50 1.00 1.50 2.00 3.00 4.00 5.00 Macrophage proliferation (%) HS1200 (%) * * * * * * * * ANNEX II LORENA GARCÍA DEL RÍO 208 List of articles: Title: Year: Journal: Volume, pages/ article number: PhD candidate contribution: Quality indexes: 1. Chapter I was published as research article. 2. Chapter III was published as research article 1. “Design of novel orotransmucosal vaccinedelivery platforms using artificial intelligence”. 2. “New tools to design smart thermosensitive hydrogels for protein rectal delivery in IBD”. 1.2021. 2.2020. 1. European Journal of Pharmaceutics and Biopharmaceutics. 2. Materials Science & Engineering C. 1. Volume 159; pages 36-43. 2. Volume 106; article number 110252. 1. The contribution of the PhD candidate to this publication consisted in work planning and data acquisition, except in the case of proliferation assays in human macrophages. These data were obtained by a contributing author. Besides, the candidate was also responsible for data analysis and writing of the original draft. 2.The contribution of the PhD candidate to this publication consisted in work planning; data acquisition; analysis and writing of the original draft. 1. The journal in which Chapter I was published presented an Impact Factor of 4.604 in 2019, belonging to quartile 1 in “Pharmacology and Pharmacy” (rank 40/271) and “Pharmacology & Toxicology” (rank 26/281) categories. 2. The journal in which Chapter III was published displayed an Impact Factor of 5.880 in 2019, belonging to quartile 1 in “Materials Science, Biomaterials” (rank 8/38) and “Materials Science” (rank 36/390) categories. Statements: conflicts of interests, image use, cell culture, in vivo assays and published content 209 Ethical considerations: Chapters I and III have been published as journal articles, edited by Elsevier. In both cases appropriate permission has been requested, and written authorization has been obtained. CHECKLISTS AND PERMISSIONS Checklists and Permissions 213 Checklist for statistical analysis. Yes/No Type and applicability of test used Comparisons of interest are clearly defined Yes Name of tests applied are clearly stated. Yes All statistical methods identified unambiguously. Yes Justification for use of test is given. Yes Data meet all assumptions of tests applied (non-normal data sets, small sample sizes) Yes Adjustments made for multiple testing is explained. N/A Details about the test n is reported at the start of the study and for each analysis thereafter. Yes Sample size calculation (or justification) is given. Yes Alpha level is given for all statistical tests. Yes Tests are clearly identified as one or two-tailed. Yes Randomization procedures or other ways to eliminate bias in sampling are described. N/A Summary of descriptive statistics n for each data set is clearly stated Yes A clearly labelled measure of centre (e.g. mean or median) is given Yes A clearly labelled measure of variability (e.g. standard deviation, range, percentiles) is given. Yes All numbers following a ± sign are identified as standard errors (s.e.m.) or standard deviations (s.d.). Yes Extras Any unusual or complex statistical methods are clearly defined and explained. Yes Any data exclusions are stated and explained. N/A Any discrepancies in the value of n between analyses are clearly explained and justified. Yes Data transformations (logarithmic, ….) are explained Yes Graphs Any distorted effect sizes (e.g. by truncation of y axis) are clearly labelled and justified. Yes Error bars in graphs, or confidence intervals, are included, or their absence is explained. Yes PhD Student signature LORENA GARCÍA DEL RÍO 214 Checklist for Thesis that include experimental animals. EXPERIMENTAL ANIMALS ARRIVE. Yes/No/NA page Title Yes Provide as accurate and concise a description of the content of the article as possible. 97; 157 Abstract Yes Provide an accurate summary of the background, research objectives, including details of the species or strain of animal used, key methods, principal findings and conclusions of the study. 99; 159 Background Yes Provide an accurate summary of the background, research objectives, including details of the species or strain of animal used, key methods, principal findings and conclusions of the study. 101; 161 Yes Explain how and why the animal species and model being used can address the scientific objectives and, where appropriate, the study’s relevance to human biology. 102; 162 Objectives Yes Clearly describe the primary and any secondary objectives of the study, or specific hypotheses being tested. 102; 162 Methods Ethical statement Yes Indicate the nature of the ethical review permissions, relevant licenses, and national or institutional guidelines for the care and use of animals, that cover the research. 108;165 Study design Yes Number of experimental and control groups 108; 165 Yes Steps taken to minimize the effects of subjective bias when allocating animals to treatment (e.g. randomization procedure) and when assessing results (e.g. if done, describe who was blinded and when). 167 Yes The experimental unit (e.g. a single animal, group or cage of animals). A time-line diagram or flow chart can be useful to illustrate how complex study designs were carried out. 109; 166 Experimental procedures Yes How (e.g. drug formulation and dose, site and route of administration, anesthesia and analgesia used [including monitoring], surgical procedure, method of euthanasia). Provide details of any specialist equipment used, including supplier(s). 108; 166 N/A When (e.g. time of day). N/A Where (e.g. home cage, laboratory, water maze). Yes Why (e.g. rationale for choice of specific anesthetic, route of administration, drug dose used). 108; 166 Experimental animals Yes Provide details of the animals used, including species, strain, sex, developmental stage (e.g. mean or median age plus age range) and weight (e.g. mean or median weight plus weight range). 108;166 Yes Provide further relevant information such as the source of animals, international strain nomenclature, genetic modification status (e.g. knock-out or transgenic), genotype, health/immune status, drug or test naïve, previous procedures, etc. 108; 166 Checklists and Permissions 215 Housing and husbandry N/A Housing (type of facility e.g. specific pathogen free [SPF]; type of cage or housing; bedding material; number of cage companions; tank shape and material etc. for fish). Yes Husbandry conditions (e.g. breeding program, light/dark cycle, temperature, quality of water etc for fish, type of food, access to food and water, environmental enrichment). 165 Yes Welfare-related assessments and interventions that were carried out prior to, during, or after the experiment. 170 Sample size Yes Specify the total number of animals used in each experiment, and the number of animals in each experimental group. 108; 166 N/A Explain how the number of animals was arrived at. Provide details of any sample size calculation used. N/A Indicate the number of independent replications of each experiment, if relevant. Allocating animals to experimental groups N/A Indicate the number of independent replications of each experiment, if relevant. N/A Describe the order in which the animals in the different experimental groups were treated and assessed. Experimental outcomes Yes Clearly define the primary and secondary experimental outcomes assessed (e.g. cell death, molecular markers, behavioral changes). Statistical methods Yes Provide details of the statistical methods used for each analysis. 111; 168 Yes Specify the unit of analysis for each dataset (e.g. single animal, group of animals, single neuron). 112; 169 N/A Describe any methods used to assess whether the data met the assumptions of the statistical approach. Results and discussion Basal data Yes For each experimental group, report relevant characteristics and health status of animals (e.g. weight, microbiological status, and drug or test naïve) prior to treatment or testing (this information can often be tabulated). 171-177 Numbers analyzed Yes Report the number of animals in each group included in each analysis. Report absolute numbers (e.g. 10/20, not 50%). 108; 166 N/A If any animals or data were not included in the analysis, explain why. Outcomes and estimation Yes Report the results for each analysis carried out, with a measure of precision (e.g. standard error or confidence interval). 113; 115; 171-177 Adverse events N/A Give details of all important adverse events in each experimental group. N/A Describe any modifications to the experimental protocols made to reduce adverse events. Interpretation/scientific implications Yes Interpret the results, taking into account the study objectives and 115; 177 LORENA GARCÍA DEL RÍO 216 hypotheses, current theory and other relevant studies in the literature. Yes Comment on the study limitations including any potential sources of bias, any limitations of the animal model, and the imprecision associated with the results. 118; 180 N/A Describe any implications of your experimental methods or findings for the replacement, refinement or reduction (the 3Rs) of the use of animals in research. Generalizability/translation N/A Comment on whether, and how, the findings of this study are likely to translate to other species or systems, including any relevance to human biology. Funding No List all funding sources (including grant number) and the role of the funder(s) in the study. Based on The ARRIVE guidelines: Animal Research: Reporting of In Vivo Experiments. PhD Student signature Checklists and Permissions 217 Checklist for recommendations of Thesis EDI saúde. GENERAL Yes-NoN/A Page For all Thesis Yes Declaration of potential conflicts of interests 207 Yes Declaration on the origin and copyright status of non original figures, with permission if necessary. Include them in the text of each figure 207 Yes Checklist of statistics adequacy if no other checklists apply. 213 For Thesis involving human experimentation, human samples, or personal data. Yes Declaration on approval by the research ethics committee. 219 Yes Code number of the study. 219; 220 No Copy of ethics report N/A Declaration that data are based on anonymous information, and no approval of the ethics committee is needed. N/A If it is an observational study, STROBE checklist. For Thesis that include a clinical assay N/A Declaration of its authorization by the Agencia Española de Medicamentos y productos sanitarios. N/A Copy of the authorization N/A CONSORT Checklist For Thesis that use embryonic or induced human stem cells Yes Declaration on its authorization 219 Yes Reference of the authorization 219 Yes Copy of the authorization 219 For Thesis that include animal experimentation Yes Declaration of its authorization 218; 220 Yes Code number of the authorization of the animal experimentation Project. 218; 220 Yes Register number of the centro de usuario autorizado if experiments were made in Spain 220 N/A Copy of the capacitation certificate if the experiments were made by the Thesis autor. Yes Person, company or service that performed the experiments if applicable. 218; 220 Yes ARRIVE Checklist 214 PhD Student signature