Microgels are solvent-swollen cross-linked gel particles with sub-micron diameters and have been widely investigated for drug delivery applications. Thermoresponsive microgels based on poly(N-isopropylacrylamide) (PNIPAM) have attracted particular attention given their potential to enable pulsatile or environment-specific drug release. However, current methods to make thermoresponsive microgels yield functionally non-degradable materials, significantly limiting their utility in vivo. Herein, hydrazone chemistry was applied to cross-link hydrazide and aldehyde-functionalized precursor polymers together to form degradable PNIPAM microgels on different length scales that enable potential use of thermoresponsive microgels in vivo in a way not currently possible. For micron-scale microgels, microfluidics was employed to create monodisperse microgels between 30-90 m. For nano-scale microgels, a temperature-driven aggregation/self-assembly technique was developed that resulted in the formation of microgels with sizes between 200-300 nm. In either case, the microgels can be slowly degraded through hydrazone hydrolysis. Functionalized microgels can be made by incorporating pH-responsive 2-dimethylaminoethylmethacrylate (DMAEMA) or glucose-responsive phenylboronic acid in the precursor polymers. The potential utility of degradable microgels in drug delivery was studied using in situ gelling microgel-hydrogel nanocomposites. Changing the microgel cross-link density and whether or not the microgels were physically entrapped or covalently cross-linked to the bulk hydrogel matrix resulted in significant changes in drug release kinetics, with burst release particularly mitigated by increasing the cross-link density of the microgels. Microgels made via microfluidics were then utilized to make fully degradable microgel-hydrogel composites consisting of chemically identical gel chemistries on both the bulk and micro length scales. Carbohydrates (carboxymethyl cellulose and dextran) and PNIPAM gel phases were oriented in different relative geometries to examine how the phase distribution impacted drug release. Results suggest that drug release can be controlled through the selection of polymer type of each phase, with the deswelling phase transitions of PNIPAM playing a particularly large role in slowing release of the drug. / Thesis / Doctor of Philosophy (PhD) / Microgels are solvent-swollen gel particles that have sub-micron diameters and have been widely investigated for a variety of biomedical applications. Temperature-responsive microgels based on poly(N-isopropylacrylamide) (PNIPAM) hold particular promise given that they can swell and deswell in response to changes in temperature, enabling pulsatile or environment-specific release of a drug. However, current thermoresponsive microgels are not degradable and therefore have limited utility in the body. In this thesis, degradable temperature-responsive microgels were developed on two length scales (micron and nano-sized) to enable their ultimate use in the body. Microgels responsive to changes in solution pH or the presence of glucose (both clinically-relevant stimuli) were made using similar techniques. Combinations of these microgels with injectable hydrogels enabled tuning of the rate of drug release by changing physical microgel and/or hydrogel, as investigated both experimentally and theoretically. The research conducted thus has the potential to impact clinical drug delivery vehicle design.

<p> Aqueous ferrofluid has been prepared via precipitation of iron oxide into a polysaccharide gel matrix followed by degradation of the polymer to form a stable magnetic colloid. Nanocrystalline particles of iron oxide were formed in an alginate network by the alkaline hydrolysis and oxidation of the crosslinking agent, Fe2+, used to bind the linear polysaccharide chains. Methanol was used to inhibit the degradation of alginate by Fe2+ and oxygen during the precipitation and growth of iron oxide particles. In addition, the structural integrity of the gel was maintained in part by interaction between the iron oxide particulate and the alginate matrix. Controlled chemical degradation of the matrix resulted in a aqueous suspension of alginate-stabilized magnetic iron oxide particles. The resulting fluid is orange-brown in color, optically transparent, superparamagnetic and stable between 2.8<pH<10 </p> <p> The magnetic gels were isolated as 2mm beads containing ~2-20 mmole Fe. X-ray and electron diffraction patterns of the composite correspond to maghemite (γ-Fe2O3) and/or magnetite (Fe3O4). At room temperature, the composite material is superparamagnetic with saturation magnetizations in excess of 20 emu g^-1 at 30kOe. TEM photomicrographs of sectioned beads and of the magnetic fluid revealed the presence of spherical nanocrystalline oxide particles with diameters ranging from 3 nm to 6 nm. The iron oxide-alginate colloid has a diameter of 54nm with an average zeta potential of -51.6 mV.</p> / Thesis / Master of Science (MSc)

The capability of precisely controlling the kinetics of therapeutic delivery at the optimal location and rate for a given patient would have great potential to improve health and well-being in a range of current drug therapies (insulin, chemotherapeutics, vaccines, etc.). Indeed, if successfully developed, locally administered injectable drug delivery vehicles capable of remotely-triggered release would be the gold standard for many treatments. Multiple injectable nanocomposites have been investigated for this purpose that are generally comprised of a thermosensitive polymeric material and superparamagnetic iron oxide nanoparticles (SPIONs). SPIONs generate heat when exposed remote alternating magnetic fields (AMFs), and the transfer of this heat to thermosensitive polymers can be used to control the release of therapeutics. Ideally, these systems would be capable of returning to their original state and basal release rate when the external AMF trigger is removed. Several novel injectable nanocomposite materials that explore interactions between SPIONs and thermosensitive polymers to mediate drug release, from the macroscale to the nanoscale, were developed and demonstrated to be capable of remotely-triggered, AMF-mediated enhanced release. The macroscale magnetic nanocomposites have thermosensitive hydrogel and/or microgel components that regulate release based on the heat produced from SPIONs in response to an external AMF. On the millimeter-scale, a microinjection system capable of producing thermosensitive hydrogel beads that could potentially incorporate SPIONs is described. On the nanoscale, nanoparticles with a glass transition temperature and thermosensitive microgels are combined with SPIONs and investigated for their remote, AMF-mediated release characteristics. The engineered macroscale and nanoscale systems are capable of up to ~4:1 and ~7:1 enhancements in release due to an AMF application, respectively, compared to the basal release rate. Collectively, these nanocomposites represent a promising stride towards improved remote-actuation of drug release and a stepping stone for future attempts at precisely controlling the site and kinetics of drug release. / Thesis / Doctor of Philosophy (PhD) / This thesis focuses on the development of nanocomposite materials that can be injected into a specific location in the body and deliver therapeutic drugs by a remote-controlled process. These nanocomposites are composed of magnetic particles and polymers that respond to changes in temperature. The combination of these materials results in nanocomposites that can change their properties in response to specific magnetic fields to switch from releasing drug slowly (or not at all) to releasing drug quickly on demand. The changes are fully reversible and solely depend on whether the external magnetic field is switched on or off. These novel systems offer an alternative to therapies that require frequent injections, such as insulin for diabetes, or therapies that need the drug to be released in very precise locations, such as cancer treatments, and could improve the safety, reduce the risk of side effects, and lower the cost of many medical treatments.

Traditional methods for dermal wound closure such as sutures and staples are invasive and can result in soft tissue trauma, increasing the likelihood of localized inflammation and infections. Alternately, while tissue adhesive alternatives can effectively seal and adhere to the wounds, they can also present safety concerns relating to immunogenic responses and tissue toxicity. Herein, we fabricate injectable, adhesive, and cytocompatible poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)-dopamine (DA) hydrogels co-crosslinked via hydrazone and self-polymerized dopamine crosslinks that exhibit high water retention, improved tissue adhesiveness, and effective tissue regeneration properties. POEGMA-DA hydrogels exhibit independently tunable gelation properties based on their dual crosslinking mechanism, allowing for gelation as fast as 24 s (allowing for injection and rapid filling of irregularly-shaped wounds) while achieving relevant compressive moduli of up to 37 kPa and in vitro skin adhesion strengths of up to 1.2 kPa. The POEGMA-DA hydrogels induced no significant inflammation while demonstrating high interfacial adhesiveness in a stented skin excisional mouse model, enabling efficient dermal tissue regeneration by supporting collagen remodelling and enabling the regeneration of hair follicles, sebaceous glands, and blood vessels at the excision site over the 14-day study timeline. As such, injectable POEGMA-DA hydrogels represent a relevant non-toxic and adhesive alternative wound closure system for treating deep dermal wounds. / Thesis / Master of Applied Science (MASc) / Effective wound healing and subsequent tissue regeneration after a physical injury requires a moist sterile environment, the presence of oxygen, nutrients and enzymes, an efficient blood supply to the wound site, and a controlled inflammatory response to initiate the healing process. External methods of closing the wound to prevent infection aid in faster healing like sutures, staples, and liquid sealants which can result in infections and/or the stimulation of an inflammatory response that can hinder tissue restoration. Hydrogels, water-swellable polymer networks, represent an alternative solution that can both suppress infection while simultaneously promoting wound healing. Hydrogels have a similar structure to soft tissues like skin and can thus provide a supportive environment for cells to promote tissue regeneration and restore tissue structure and function. The swelling of hydrogels in water is highly beneficial for providing moisture at the wound site; however, this high degree of water retention also means they have a hard time sticking to tissues. To address this challenge, hydrogels can be modified with a component naturally derived from marine mussels that allows them to stick to their wet habitats, helping hydrogels to stick to the wound site while healing. In this thesis, mussel-inspired hydrogels are designed and can spontaneously gel and stick to a wound site to accelerate the restoration of the structure and function of skin. These biodegradable and injectable hydrogels are effective in accelerating wound closure with minimal evidence of scarring while suppressing negative inflammatory reactions and restoring the structure of skin by promoting the regeneration of hair follicles, sebaceous glands and blood vessels.

The extracellular matrix (ECM) is the highly hydrated, protein- and glycosaminoglycan- (GAG) based cell environment that provides cell-instructive cues like the mechanical stabilization of the cells and transmission of biochemical and physical signals. To biochemically and mechanically mimic the ECM, hydrogels with the highly negatively charged GAG heparin in interplay with a stabilizing polymer network are of high interest in biomaterial engineering. The application as cell-instructive materials allows for controlling transport processes of signaling molecules within the matrices, cell growth and differentiation behavior, and cellular fate decisions. In particular, heparin-based biomaterials enable targeted sequestration of signaling molecules on the one hand, but also sustained delivery of them with a lower necessary amount to be used, in contrast to the discontinuous application of solutes. In addition, heparin-based biomaterials can protect the loaded cargo from enzymatic degradation and conformational changes.[1]–[3] The affinity to signaling molecules as key feature provides the potential for applications in wound healing and tissue regeneration. Synthetic sulfonated polymers (SSPs) as synthetic heparin analogs can address multiple drawbacks of native heparin, such as its heterogeneous chemical structure and the potential risk of viral contamination from the animal isolation source.[4],[5] Due to a large number of molecular design opportunities in particular the degree of sulfation, sulfate volume concentration, sulfate or sulfonate nature, distance of the sulf(on)ate from the backbone, and hydrophobicity of the polymers, biochemical processes may be controlled in a targeted manner. The chemical possibilities for forming a hydrogel network based on SSPs are far more diverse with synthetic, freely designable polymers to achieve a targeted structure and chemical nature of the network. Here, the aim was to introduce a library of SSPs to replace heparin in fully synthetic hydrogels capable of modulating cell-instructive cues such as soluble factor signaling, adhesiveness, and growth behavior of integrated cells. Accordingly, a library of systematically varied SSPs differing in degree of sulfation, sulfate or sulfonate conjugation, hydrophobicity, and sulf(on)ate distance to the backbone have been synthesized from by polymer analog reaction of various sulf(on)ated amines with a polyacrylate (15 kDa, sodium salt) as the polymeric backbone. The polymers have been thoroughly characterized by proton nuclear magnetic resonance (1H-NMR), Fourier-transform infrared spectroscopy (FTIR), asymmetric flow field flow fractionation (AF4) coupled light scattering analysis, and microscale thermophoresis (MST) for their molecular composition, stability in aqueous solution, conformation, and interaction with a chosen signal molecule. The affinity of the very stable coiled polymers under physiological conditions to signaling molecules depends mainly on the degree of sulfation, sulfate or sulfonate nature, and hydrophobicity. The SSPs are crosslinked with 4-arm star-shaped poly(ethylene glycol) (starPEG) either directly to form amide-crosslinked hydrogels or by pre-functionalization via Michael-type addition to prepare cell-instructive hydrogels, each with graded mechanical properties. The affinity of these hydrogels for various signaling molecules can be quantified compared to heparin-based ones and attributed to the influence of the degree of sulfation, sulfate volume concentration, sulfate or sulfonate nature, and hydrophobicity. The potential of SSPs in functional 3D tissue cultures could be confirmed by renal morphogenesis and neural network formation in the corresponding hydrogels by collaborators. Further on, the synthesis procedure of hydrogel precursors has been transferred to fully automated procedures. Because standardized production of cell-instructive hydrogels at low compositional and batch-to-batch variation and material compliance can benefit from high-throughput synthesis and liquid handling robots. An automated multistage workflow was developed to synthesize hydrogel precursors, carry out hydrogel formation, and execute cell culture experiments with cells embedded in the hydrogels. The protocol combines two robotic liquid handling systems and a microscope for automated sample imaging and cell analysis. The customized heparin and SSP maleimidation procedures, including temperature-regulated synthesis, purification, and aliquotation, were implemented on a customized liquid-handling robot. The resulting hydrogel precursors were analyzed for their maleimide conjugation efficiency and purity by 1H-NMR and conductivity measurements and for their hydrogel formation ability. This automated synthesis can ensure the quality and production of good manufacturing practice (GMP)-compliant hydrogel materials. Automated SSP hydrogel preparation, cell culture, and analysis can further promote combinatorial approaches to biomedical applications of cell-instructive materials. References [1] Lohmann, N.; Schirmer, L.; Atallah, P.; Wandel, E.; Ferrer, R. A.; Werner, C et al. Glycosaminoglycan-Based Hydrogels Capture Inflammatory Chemokines and Rescue Defective Wound Healing in Mice. Sci. Transl. Med. 2017, 9 (386), 1–12. [2] Schirmer, L.; Atallah, P.; Werner, C.; Freudenberg, U. StarPEG-Heparin Hydrogels to Protect and Sustainably Deliver IL-4. Adv. Healthc. Mater. 2016, 5 (24), 3157–3164. [3] Liang, Y.; Kiick, K. L. Heparin-Functionalized Polymeric Biomaterials in Tissue Engineering and Drug Delivery Applications. Acta Biomater. 2014, 10 (4), 1588–1600. [4] Blossom, D. B.; Kallen, A. J.; Patel, P. R.; Elward, A.; Robinson, L.; Gao, G. et al. Outbreak of Adverse Reactions Associated with Contaminated Heparin. N. Engl. J. Med. 2008, 359 (25), 2674–2684. [5] Hirsh, J.; Dalen, J. E.; Anderson, D. R.; Poller, L.; Bussey, H.; Ansell, J. et al. Oral Anticoagulants. Chest 1998, 114 (5), 445S-469S.

Tesis por compendio / [ES] La tesis doctoral se centra en el desarrollo de un hidrogel sensible a analitos, funcionalizado con sondas de ADN, con estructura difractiva como transductor óptico para aplicaciones de diagnóstico in vitro. El primer capítulo incluye una visión general de los diferentes conceptos relacionados con el biosensado, los desarrollos recientes en el mercado del diagnóstico in vitro y, en particular, los biosensores de ADN. Además, se presenta la síntesis y caracterización de hidrogeles, su papel como matriz de soporte en biosensado y las estrategias de inmovilización. Por último, se explican los conceptos básicos de la holografía como nueva estrategia de detección y el papel de las diferentes redes de difracción en la biosensación. A continuación, en el Capítulo 2, se discuten los objetivos de este proyecto. El objetivo de esta investigación es desarrollar hidrogeles que incorporen sondas de ADN y dotarlas de una estructura difractiva para que actúen como transductores ópticos sin etiquetas. Se consideran dos tipos de estructuras difractivas: redes holográficas de relieve superficial (SRG) y redes de transmisión de volumen (VTG). La fase inicial de este trabajo se centró en la optimización de hidrogeles, ajustando su composición para que actuaran como biosensores holográficos. Se seleccionaron acrilamida y bisacrilamida para la preparación del hidrogel mediante reacción de polimerización por radicales libres. Además, para introducir la respuesta del analito en la red de hidrogeles 3D, hubo que investigar y poner a punto diferentes estrategias de inmovilización de biorreceptores. En el capítulo 3, la estrategia optimizada consiste en incorporar directamente sondas de ADN modificadas con acridita mediante copolimerización con monómeros de acrilamida durante la formación del hidrogel. Los hidrogeles funcionalizados con ADN se caracterizaron mediante imágenes de fluorescencia y se exploró su versatilidad mediante la fabricación de microarrays. Por último, el hidrogel optimizado sensible a los analitos se utilizó como plataforma para la preparación de SRG. El capítulo 4 describe otro enfoque adoptado para la funcionalización del hidrogel con sondas de ADN. Se añadió un comonómero de acrilato de propargilo al hidrogel de acrilamida, con el fin de introducir la presencia de residuos alcínicos y facilitar una mayor incorporación de las sondas de ADN. Las sondas de ADN utilizadas tenían grupos terminales tiol y se incorporaron mediante química de clic tiol-eno/tiol-yo, debido a la presencia de enlaces C-C dobles y triples. Con esta estrategia, se demostraron dos enfoques de inmovilización de sondas de ADN: durante y después de la síntesis del hidrogel. Los resultados preliminares mostraron que los SRGs tienen potencial para detectar directamente la hibridación de oligonucleótidos en un formato libre de etiquetas. En el capítulo 5, se optimizó el proceso de grabación de VTGs no inclinados en capas de hidrogel para mejorar el rendimiento del transductor. Tras una cuidadosa evaluación de los parámetros de grabación holográfica, las composiciones de las soluciones de incubación y los tiempos de incubación, las estructuras VTG se grabaron con una buena reproducibilidad, logrando una excelente eficiencia de difracción. Además, se estudió su estabilidad en agua para bioensayos. Por último, se observó que los VTG, modificados con oligonucleótidos, respondían selectivamente hibridándose sólo con la diana complementaria, a la vez que conservaban sus propiedades de difracción. El trabajo de investigación demostró la viabilidad de utilizar redes difractivas en capas de hidrogel como biosensores libres de etiquetas, capaces de detectar sondas de ADN, complementarias a la secuencia inmovilizada, en un medio acuoso. Por último, en el capítulo 6, se analizan comparativamente el rendimiento y la aplicabilidad de los distintos enfoques estudiados y se discuten las perspectivas futuras de los hidrogeles de ácidos nucleicos para la detección holográfica. / [CA] La tesi doctoral se centra en el desenvolupament d'un hidrogel sensible a anàlits, funcionalitzat amb sondes d'ADN, amb estructura difractiva com a transductor òptic per a aplicacions de diagnòstic in vitro. El primer capítol inclou una visió general dels diferents conceptes relacionats amb el biosensado, els desenvolupaments recents en el mercat del diagnòstic in vitro i, en particular, els biosensores d'ADN. A més, es presenta la síntesi i caracterització d'hidrogels, el seu paper com a matriu de suport en biosensado i les estratègies d'immobilització. Finalment, s'expliquen els conceptes bàsics de l'holografia com a nova estratègia de detecció i el paper de les diferents xarxes de difracció en la biosensación. A continuació, en el Capítol 2, es discuteixen els objectius d'este projecte. L'objectiu d'esta investigació és desenvolupar hidrogels que incorporen sondes d'ADN i dotar-les d'una estructura difractiva perquè actuen com a transductors òptics sense etiquetes. Es consideren dos tipus d'estructures difractivas: xarxes hologràfiques de relleu superficial (SRG) i xarxes de transmissió de volum (VTG). La fase inicial d'este treball es va centrar en l'optimització d'hidrogels, ajustant la seua composició perquè actuaren com biosensores hologràfics. Es van seleccionar acrilamida I bisacrilamida per a la preparació de l'hidrogel mitjançant reacció de polimerització per radicals lliures. A més, per a introduir la resposta de l'anàlit en la xarxa d'hidrogels 3D, va caldre investigar i posar a punt diferents estratègies d'immobilització de biorreceptores. En el capítol 3, l'estratègia optimitzada consisteix a incorporar directament sondes d'ADN modificades amb acridita mitjançant copolimerización amb monòmers d'acrilamida durant la formació de l'hidrogel. Els hidrogels funcionalitzats amb ADN es van caracteritzar mitjançant imatges de fluorescència i es va explorar la seua versatilitat mitjançant la fabricació de bioxips. Finalment, l'hidrogel optimitzat sensible als anàlits es va utilitzar com a plataforma per a la preparació de SRG. El capítol 4 descriu un altre enfocament adoptat per a la funcionalització de l'hidrogel amb sondes d'ADN. Es va afegir un comonómero de acrilato de propargilo a l'hidrogel d'acrilamida, amb la finalitat d'introduir la presència de residus alcínicos i facilitar una major incorporació de les sondes d'ADN. Les sondes d'ADN utilitzades tenien grups terminals tiol i es van incorporar mitjançant química de clic tiol-eno/tiol-ino, a causa de la presència d'enllaços C-C dobles i triples. Amb esta estratègia, es van demostrar dos enfocaments d'immobilització de sondes d'ADN: durant i després de la síntesi de l'hidrogel. Els resultats preliminars van mostrar que els SRGs tenen potencial per a detectar directament la hibridació de oligonucleótidos en un format lliure d'etiquetes. En el capítol 5, es va optimitzar el procés de gravació de VTGs no inclinats en capes d'hidrogel per a millorar el rendiment del transductor. Després d'una acurada avaluació dels paràmetres de gravació hologràfica, les composicions de les solucions d'incubació i els temps d'incubació, les estructures VTG es van gravar amb una bona reproducibilidad, aconseguint una excel·lent eficiència de difracció. A més, es va estudiar la seua estabilitat en aigua per a bioensayos. Finalment, es va observar que els VTG, modificats amb oligonucleótidos, responien selectivament hibridant-se només amb la diana complementària, alhora que conservaven les seues propietats de difracció. El treball de recerca va demostrar la viabilitat d'utilitzar xarxes difractivas en capes d'hidrogel com biosensores lliures d'etiquetes, capaces de detectar sondes d'ADN, complementàries a la seqüència immobilitzada, en un medi aquós. Finalment, en el capítol 6, s'analitzen comparativament el rendiment i l'aplicabilitat dels diferents enfocaments estudiats i es discuteixen les perspectives futures dels hidrogels d'àcids nucleics per a la detecció hologràfica. / [EN] The PhD thesis focuses on the development of an analyte-sensitive hydrogel, functionalised with DNA probes, with a diffractive structure as an optical transducer for in vitro diagnostic applications. The first chapter includes an overview of the different concepts related to biosensing, recent developments in the in vitro diagnostics market and, in particular, DNA biosensors. Furthermore, the synthesis and characterisation of hydrogels, their role as a support matrix in biosensing and immobilisation strategies are presented. Finally, the basic concepts of holography as a new detection strategy and the role of different diffraction gratings in biosensing are explained. Then, in Chapter 2, the objectives of this project are discussed. The aim of this research is to develop hydrogels that incorporate DNA probes and provide them with a diffractive structure to act as label-free optical transducers. Two types of diffractive structures are considered: surface-relief holographic gratings (SRGs) and volume transmission gratings (VTGs). The initial phase of this work focused on the optimisation of hydrogels, adjusting their composition to act as holographic biosensors. Acrylamide and bisacrylamide were selected for hydrogel preparation by free radical polymerisation reaction. Furthermore, in order to introduce the analyte response into the 3D hydrogel network, different bioreceptor immobilisation strategies had to be investigated and fine-tuned. In chapter 3, the optimised strategy is to directly incorporate acridite-modified DNA probes by copolymerisation with acrylamide monomers during hydrogel formation. The DNA-functionalised hydrogels were characterised by fluorescence imaging and their versatility was explored by microarray fabrication. Finally, the optimised analyte-responsive hydrogel was used as a platform for SRG preparation. Chapter 4 describes another approach adopted for functionalisation of the hydrogel with DNA probes. A propargyl acrylate comonomer was added to the acrylamide hydrogel in order to introduce the presence of alkyl residues and facilitate further incorporation of the DNA probes. The DNA probes used had thiol end-groups and were incorporated by thiol-ene/thiol-yo click chemistry, due to the presence of double and triple C-C bonds. With this strategy, two approaches to DNA probe immobilisation were demonstrated: during and after hydrogel synthesis. Preliminary results showed that SRGs have the potential to directly detect oligonucleotide hybridisation in a label-free format. In chapter 5, the recording process of unslanted VTGs in hydrogel layers was optimised to improve transducer performance. After careful evaluation of holographic recording parameters, incubation solution compositions and incubation times, the VTG structures were recorded with good reproducibility, achieving excellent diffraction efficiency. In addition, their stability in water for bioassays was studied. Finally, oligonucleotide-modified VTGs were found to respond selectively by hybridising only to the complementary target, while retaining their diffraction properties. The research work demonstrated the feasibility of using diffractive networks in hydrogel layers as label-free biosensors, capable of detecting DNA probes, complementary to the immobilised sequence, in an aqueous medium. Finally, in chapter 6, the performance and applicability of the different approaches studied are comparatively analysed and future prospects of nucleic acid hydrogels for holographic detection are discussed. / I would like to acknowledge the government of Valencia to for the PhD fellowship “Santiago Grisolia” and the BEFPI/2022 grant for a 4-months doctoral stay and also the Spanish Ministry of Economy and Competitiveness MINECO (ADBIHOL national project) for their financial support. / Zezza, P. (2023). Holographic biosensors made of DNA-functionalised hydrogels for in vitro diagnostic [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202597 / Compendio

Food insecurity is a growing issue in the United States. Iron deficiency is the most common form of nutritional deficiency in patients with endothelial dysfunction and vascular-related diseases. This preliminary study lays the groundwork for the “Nutrient deficiency-on-a-chip” model. Endothelial cells are cultured on mechanically tunable, enzymatically cross-linked gelatin and treated with deferoxamine, an iron chelator, or angiotensin II were used to simulate a nutrient deficient and diseased environment, respectively. As oxidative stress and disturbed barrier function are the most prevailing mechanism of angiotensin II and iron deficiency induced endothelial dysfunction, to test our model we investigated the changes in reactive oxygen species production and VE-cadherin expression in engineered endothelium. Both angiotensin II and deferoxamine treated engineered endothelium showed an increase in oxidative stress and disturbed barrier function. This in vitro model can be a useful tool to better understand disease mechanisms associated with nutrient deficiency and identify novel therapeutics.

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