FCT: DQ - Teses de Doutoramento
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- Advanced water-soluble soft nanomaterials encapsulating luminescent inorganic and organic emitters and hydrophobic drugs as novel nanocarriers: from bioimaging to drug delivery and environmental applicationsPublication . Duarte, Frederico Gonçalo do Vale; Lodeiro Y Espiño, Carlos; Marques, Elisabete; Cuerva de Alaíz, CristiánFluorescent molecular probes have become indispensable tools across chemical, envi- ronmental, and biomedical sciences, yet current sensing technologies often lack the sensitivity, multifunctionality, and adaptability required to address today’s analytical challenges. With in- creasing concerns regarding environmental pollutants, toxic metal exposure, and real-time di- agnostics, there is a growing demand for highly responsive, cost-effective, and versatile fluo- rescent systems capable of reliable performance across diverse settings. This doctoral thesis addresses this need by developing innovative molecular platforms that combine advanced photophysical design with practical sensing capabilities. It presents the conception, synthesis, and application of next-generation multifunctional fluorophores, show- ing how strategic chromophore engineering can improve detection accuracy, environmental responsiveness, and functional versatility. Central to this research is the construction of eighteen dansyl-based fluorophores in- tegrating tailored receptor architectures, macrocyclic frameworks, and dual-chromophoric as- semblies to enhance optical responses and enable finely tuned sensing pathways. Key advance- ments include (i) the development of solvatochromic and aggregation-induced emission (AIE) systems capable of reporting subtle changes in microenvironmental polarity and water con- tent; (ii) the design of single-chromophore and dyads that exploit ICT, PET, and CHEF mecha- nisms for selective detection of toxic ions such as Hg²⁺, Cu²⁺, and cyanide; (iii) the incorporation of disulfide (S–S) and thiol (–S–) functionalities that induce fluorescence quenching with emis- sion restored upon cleavage and serve as recognition sites for soft-soft selective Hg²⁺ detection; (iv) the design of macrocycle-based dansyl platforms that enhance light absorption and fluorescence intensity while alkyl side chains promote mesomorphic organization, improving stability in aggregated environments; (v) the fabrication of polymeric particles and films providing water stability, temperature-responsive sensing by incorporating the fluorophores which can also act as stabilizers during metallic nanoparticle synthesis; and (vi) the discovery of fluor- ophores exhibiting intrinsic antimicrobial activity, demonstrating multifunctionality beyond sensing. Overall, this thesis shows how multifunctional dansyl-based fluorophores can be rationally designed to combine tunable photophysics, selective sensing, material fabrication, and biological activity. These findings provide a versatile framework for developing next-generation fluorescent materials with applications in environmental monitoring, analytical chemistry, and biomedical diagnostics.
- Marine waste streams valorization for the production of novel bioactive 3D structures and their evaluation for potential biomedical applicationsPublication . Batista, Miguel Pedro Lopes; Bronze, Maria do Rosário; Duarte, Ana; Gaspar, FrédéricThe underutilization of marine byproducts contributes to waste accumulation and economic loss. The scientific community has recently focused on extracting valuable polymers like collagen for high-value products in food, cosmetics, and biomedical applications. However, current recovery methods are time-consuming, chemically intensive, and energy demanding. This dissertation focused on using natural deep eutectic solvents (NADES) to extract marine collagen from fish processing waste. After extraction, this work intended to process the collagen-NADES extract to develop new porous materials. Hence, the main scope of this thesis was to: i) Develop a novel extraction using NADES to intensify the conventional process and charac- terize the protein purity and structure. ii) Perform a comparative life cycle assessment (LCA) of conventional and NADES-enhanced processes. iii) Implement an unexplored route for collagen drying materials using supercritical CO2. iv) Prepare a porous material from chitosan and collagen-NADES extract loaded with hydroxy- tyrosol (HT). Results revealed that the NADES-enhanced process yielded pure type I collagen with 2.5 times higher efficiency while reducing the extraction time from 96 h to 1 h compared to the conventional approach. The LCA of both methods indicated that producing 1 kg of purified marine collagen using this NADES approach reduces 14% of the conventional process’s environmental impact. However, the LCA also showed that extraction processes involving NADES must be deeply scrutinized due to the impact of NADES component production. Drying the gelled collagen extract with supercritical CO2 was a suitable technique to produce collagen aerogels showing a relatively dense mesoporous network with a specific surface area and pore volume of 201–203 m2/g and 1.08–1.15 cm3/g, respectively. Physicochemical characterization confirmed collagen purity and that the production process does not impact protein tertiary structure. Alternatively, incorporating chitosan and HT improved freeze-dried collagen material's mechanical robustness and biological performance. HT showed a controlled release profile, up to 70% after 10 h. Both fabricated 3D structures produced from the collagen-NADES extract exhibited suitable features for potential topical biomedical applications. This dissertation contributes to the circular economy, addressing fish processing waste and the environmental footprint of collagen extraction activities. The present work also promotes biomedical innovation and the valorization of marine byproducts, showcasing the versatility of collagen materials and the therapeutic potential of marine waste.
- Development of polymeric dressings for advanced wound care based on bacterial cellulose and Fu- coPolPublication . Esmail, Asiyah; Freitas, Maria Filomena; Serra, Ana TeresaBacterial cellulose (BC) is a biopolymer characterised by high purity, mechanical re- sistance, water-holding capacity and biocompatibility, making it a strong candidate for wound dressing applications. However, high production costs and lack of intrinsic bioactivity remain major limitations. This thesis addressed these challenges by (i) producing BC from sustainable feedstocks and (ii) developing BC:FucoPol (FP) composites with bioactive properties. BC was produced from unconventional substrates, including polyethene terephthalate (PET) monomers (terephthalic acid and ethylene glycol), styrene and food waste (stale bread hydrolysate and waste apple pulp). BC production from plastic-derived monomers was feasible but yielded ≤1 g/L, requiring glucose supplementation. Food wastes performed far better: stale bread hydrolysate resulted in a production of 2.40 g/L. In comparison, waste apple pulp (APPsup) yielded up to 3.38 g/L, surpassing the production in standard glucose-supplemented Hestrin–Schramm medium (1.5-2.1 g/L). The BC obtained from APPsup also exhibited favour- able mechanical and barrier properties, making it the most promising feedstock among the tested substrates. To confer bioactivity, FP, an exopolysaccharide with antioxidant, photoprotective and wound-healing properties, was incorporated into BC through three strategies. In-situ compo- sites, obtained by adding FP to the culture medium during biosynthesis, enabled homogene- ous FP distribution and retained thermal stability (Tdeg = 317–343 °C), though structural dis- ruption occurred above 1 wt.% FP. Ex-situ Fe³⁺-crosslinked composites, produced by solvent immersion followed by ionic gelation, reinforced the membranes' mechanical performance (Storage modulus (G') up to 57.1 kPa) and promoted cell adhesion and spreading. Still, FP remained in a gelled, less diffusible state. In contrast, simple ex-situ impregnation (BC_IMFP), without Fe³⁺-induced gelation, preserved FP’s bioactivity. Although it reduced the membrane's thermal stability (276 °C vs. 340 °C), it strongly enhanced keratinocyte viability (up to 159%) and significantly improved wound closure (28.3 ± 7.5% in 24 h). Overall, this work demonstrates that BC can be sustainably produced from waste and petrochemical-derived substrates, and the resulting membranes can be functionalised with the bioactive polysaccharide FP to generate multifunctional wound dressings. Each formulation provides distinct advantages for different uses: neat BC membranes can be used for fluid man- agement, the Fe³⁺-crosslinked composites offer improved mechanical reinforcement and pro- mote cell adhesion, and the BC_IMFP displayed fast wound closure ability. These findings es- tablish BC/FP systems as versatile dressings, bridging waste valorisation to advanced thera- peutic applications.
- From Biomarkers to Transcriptomics: Assessing the Impact of PFOA, TBBPA, and EE2 on the Sentinel Species Mytilus galloprovincialisPublication . Copeto, Sandra Cristina Marinheiro Ferreira; Diniz, Mário; Motta, Carla; Silva, MarcoEmerging contaminants, particularly endocrine-disrupting chemicals (EDCs), are in- creasingly detected in marine ecosystems, food, and consumer products, posing risks to both wildlife and human health. Among them, perfluorooctanoic acid (PFOA), tetrabromobisphenol A (TBBPA), and 17α-ethinylestradiol (EE2) stand out due to their environmental persistence, bioaccumulation potential, and ability to interfere with hormonal regulation, oxidative balance, and cellular homeostasis. However, their toxicological effects on marine bivalves, key sentinel species, remain poorly understood. Mussels such as Mytilus galloprovincialis play a central ecological role and are widely used as bioindicators because of their capacity to accumulate contaminants through filter feed- ing. Assessing their biochemical and transcriptomic responses provides critical insight into the sublethal and realistic effects of EDCs at multiple levels of biological organization. This is par- ticularly relevant given the high seafood consumption in Europe, which represents a major human exposure pathway to these contaminants. Laboratory exposures of M. galloprovincialis were conducted using environmentally relevant concentrations of PFOA (1–100 μg·L⁻¹), TBBPA (1–100 μg·L⁻¹), and EE2 (10–300 ng·L⁻¹). A multi-biomarker strategy was applied to evaluate oxidative stress (SOD, CAT, GST, TAC, MDA), protein degradation (UBI), apoptosis (caspase-3), neurotoxicity (AChE), and endocrine disruption (VTG). In parallel, transcriptomic analyses were performed to identify differentially expressed genes and disrupted pathways related to stress responses, detoxification, lipid me- tabolism, and endocrine regulation. The results revealed concentration-dependent alterations in antioxidant defences and cellular homeostasis, with PFOA and TBBPA inducing oxidative stress and apoptosis, while EE2 primarily triggered estrogenic and gametogenic disruptions. Transcriptomic analysis confirmed the modulation of pathways associated with xenobiotic metabolism, energy balance, apoptosis, and non-genomic estrogen signaling. Together, these results indicate that M. gal- loprovincialis exhibits coordinated biochemical and molecular responses upon exposure to EDCs, reinforcing its relevance as a sentinel species. The study highlights potential risks of contaminant biomagnification through the food chain and provides mechanistic evidence to support ecotoxicological monitoring frameworks and regulatory measures aimed at protecting marine ecosystems and public health.
- The cephalopod skin as a source of proteins: lessons from adaptation and ecophysiologyPublication . Padrão, Inês Rodrigues; Roque, Ana Cecília; Costa, PedroColeoid cephalopods are organisms with high ecological and economical relevance, distinguished by rapid and reversible changes in body coloration, skin patterning and texture. This is achieved by the interplay between pigmentary chromatophores and structural iridophores and leucophores, forming a layered tissue that supports camouflage, signaling and environmental sensing. The skin of cuttlefish and squid have been extensively investigated at the structural and protein levels, while the molecular basis of skin function in Octopus vulgaris remains comparatively less understood. Expanding the knowledge in this area is not only relevant for evolutionary and ecological biology, but also provides a foundation for blue biotechnology, where marine- derived proteins are increasingly recognized as sustainable sources for innovation. The main goal of this thesis is to address this gap by integrating morphological, biophysical and transcriptomic approaches. Light microscopy, transmission electron microscopy (TEM) and focused ion beam-scanning electron microscopy (FIB-SEM) provided insights into the integrated function of chromatophores, iridophores and leucophores in camouflage. Skin protein content analysis and transcriptomics confirmed the presence of reflectins and crystallins, and suggested novel skin sensory roles. Secondly, a reflectin from O. vulgaris (Rov1) was identified, expressed and characterized. This protein was shown to be intrinsically disordered, aggregation-prone and thermally stable with moderate pH-responsive assembly dynamics. Lastly, comparative transcriptomics between the skin of O. vulgaris and the polar squid Galiteuthis glacialis revealed distinct strategies. Octopus skin preferred reflectins and neural associated transcripts for fast camouflage. Squid, however, relied on crystallin-like proteins, chemosensory receptors and muscle structure for transparency, environmental sensing and locomotion. By advancing the molecular and structural characterization of cephalopod skin, this work contributed with new insights into dynamic coloration and sensory integration. These findings also highlight the relevance of cephalopod-derived proteins as candidates for future exploration within blue biotechnology.
- Transdermal Delivery of Pharmaceuticals with Ionic Systems and NanomaterialsPublication . Faísca, Francisco Luís de Aragão; Branco, Luís; Lima, SofiaA administração transdérmica de fármacos constitui uma alternativa promissora às vias convencionais. No entanto, a maioria dos antibióticos, devido à sua elevada polaridade e baixa lipofilicidade, apresenta limitações significativas na capacidade de atravessar eficazmente a barreira cutânea. Esta tese explora a utilização de sais e líquidos iónicos derivados do próprio fármaco (API-OSILs) como estratégia química para melhorar a solubilidade, lipofilicidade e permeabilidade cutânea de antibióticos. Avalia-se ainda a sua integração em plataformas avançadas de administração, incluindo nanopartículas lipídicas e microagulhas dissolúveis. A cefuroxima e a estreptomicina foram selecionadas como sistemas modelo para investigar como a conjugação com iões orgânicos poderá potenciar a administração transdérmica. O trabalho está organizado em quatro estudos principais: (1) síntese e avaliação de sais de cefuroxima (CFX-OSILs), (2) desenho e triagem de sais orgânicos de estreptomicina (STP-OS), (3) formulação destes compostos em nanopartículas lipídicas sólidas e (4) incorporação em dispositivos de microagulhas dissolúveis. Para explorar o potencial da modificação iónica na melhoria da administração transdérmica de antibióticos, a tese inicia-se com a síntese de sais orgânicos de cefuroxima utilizando catiões de piridínio e imidazólio. Estes sais aumentaram a solubilidade original, até 200 vezes superior à do fármaco de base a 37 °C, e modificaram o comportamento de partição, sendo que catiões mais lipofílicos aumentaram a afinidade pela fase orgânica. Entre eles, [PyC10Py][CFX]2 manteve atividade antibacteriana, enquanto outros apresentaram eficácia reduzida. Os ensaios de citotoxicidade revelaram maior toxicidade apenas em sais contendo cadeias longas de alquilo. Embora não fossem candidatos finais à administração transdérmica, este estudo forneceu contributos metodológicos relevantes para a modificação iónica e estabeleceu uma base para aplicar esta estratégia a antibióticos mais desafiantes e clinicamente relevantes. Dando seguimento a esta abordagem, o segundo estudo aplicou uma estratégia semelhante de modificação iónica à estreptomicina, um antibiótico aminoglicosídeo particularmente desafiante. Foram sintetizados oito sais orgânicos de estreptomicina (STP-OS), contendo contra-iões sulfonato e carboxilato, que originaram sólidos estáveis e foram caracterizados sistematicamente. Em estudos de permeação através de bicamadas fosfolipídicas, [STPH3][p-TolSO3]3 apresentou a maior permeabilidade cutânea, [STPH3][C6SO3]3 exibiu um equilíbrio entre permeabilidade e solubilidade, enquanto [STPH3][GlyCOO]3 mostrou maior solubilidade mas permeação negligenciável. Os ensaios antimicrobianos confirmaram a manutenção da atividade contra estirpes suscetíveis. Em conjunto, estes resultados demonstram que a modificação iónica pode ser uma ferramenta versátil para ajustar a solubilidade, lipofilicidade e interação com membranas da estreptomicina, estabelecendo uma base química para a sua incorporação em sistemas avançados de administração transdérmica. Com base nos resultados promissores da modificação iónica, o terceiro estudo investigou a incorporação de STP-OS selecionados: [STPH3][p-TolSO3]3, [STPH3][C6SO3]3 e [STPH3][GlyCOO]3, em paralelo com o sal convencional de sulfato, em nanopartículas lipídicas sólidas (NLS) utilizando Softisan® como matriz lipídica. As nanopartículas resultantes apresentaram cerca de 150 nm de diâmetro, índices de polidispersidade baixos (~0,2) e potenciais zeta negativos (~–30 mV), indicadores de estabilidade coloidal. As eficiências de encapsulação variaram entre 35 e 62%, com cargas de fármaco entre 4-6%, demonstrando que todos os sais eram globalmente compatíveis com o veículo lipídico. Géis liofilizados obtidos a partir destas NLS mostraram-se adequados para aplicação dérmica, com manuseamento e consistência favoráveis. Os ensaios de CIM revelaram maior potência antimicrobiana das formulações em NLS comparativamente aos sais livres, reforçando o valor da encapsulação em nanopartículas. Importa salientar que a formulação bem-sucedida de sais com perfis distintos de solubilidade e permeabilidade demonstrou que a identidade do OSIL não comprometeu a formulação, permitindo ainda afinar o comportamento de administração. O quarto e último estudo incorporou os mesmos três STP-OS em microdispositivos dissolúveis (MAPs) compostos por PVP/PVA. O sal de sulfato convencional apresentou menor solubilidade na matriz polimérica, resultando em menor carga de fármaco quando comparado com os dispositivos contendo STP-OS. Apesar destas diferenças, todos os MAPs apresentaram excelentes propriedades mecânicas, com >99% de inserção confirmada por tomografia de coerência ótica e microscopia. Estudos ex vivo em pele de leitão de espessura total revelaram que, embora a carga total de fármaco variasse, os perfis de libertação e deposição foram comparáveis entre formulações. Curiosamente, os OSILs apresentaram ligeiramente maior retenção nas camadas cutâneas. Estes resultados evidenciam a importância da solubilidade na matriz de formulação e demonstram como a modificação iónica pode expandir as opções de formulação em sistemas de administração transdérmica. Em conjunto, os quatro estudos desta tese demonstram que os API-OSILs representam uma plataforma química para modular a solubilidade, lipofilicidade, permeabilidade e perfis de administração de antibióticos. Além disso, estas modificações podem ser com sucesso transpostas para sistemas avançados de administração, como nanopartículas lipídicas e microagulhas dissolúveis. Embora a extensão da melhoria dependa da molécula, como observado na modulação limitada da estreptomicina altamente hidrofílica, os resultados fornecem uma prova de conceito de que a modificação iónica constitui uma estratégia viável e adaptável para potenciar e otimizar a administração transdérmica de fármacos.
- Development of Active Packaging Based on Cynara cardunculus L. Extract to Extend Food's Shelf LifePublication . Barbosa, Cássia Helena Vieira; Silva, Ana Teresa; Fernando, Ana Luísa; Vilarinho, FernandaCardoon (Cynara cardunculus L.) is a plant valued for its roles in human food, biomass production, and as a vegetable coagulant in cheese production. Its leaves, often discarded as by-products, exhibit significant biological activities, such as antioxidant and antimicrobial properties. This work aimed to formulate an active food film/coating based on whey protein, incorporating cardoon leaf extract to reduce cheese deterioration and extend its shelf life. The thesis assessed the extract's antioxidant, antibacterial, and antifungal capacities and evaluate the packaging’s mechanical and barrier properties. Subsequently, the effectiveness of the packaging was evaluated in terms of its ability to preserve cheese quality and increase its shelf life. This thesis demonstrated the potential of cardoon leaves as a valuable source of bioactive compounds. The dried cardoon leaves extract showed strong antioxidant properties, being rich in chlorogenic acid, apigenin and luteolin, as well significant antimicrobial activity. Cardoon leaves also represent a good source of dietary fibre, amino acids, and minerals. The incorporation of the cardoon extract, alone or in combination with lemon peel extract, into whey protein-based films improved their barrier and optical properties, although some reduction in mechanical properties was observed. The coatings and films were effective in maintaining cheese quality during storage. Coatings with only cardoon extract exhibited moderate antimicrobial activity, while the combination with lemon peel extract or natamycin enhanced microbial inhibition. The cardoon extract was also applied to poultry meat, effectively extending its shelf life by delaying lipid oxidation and microbial growth. These results support the hypothesis that cardoon leaf extracts can be valorised as an active compound in the development of sustainable packaging materials. By repurposing cheese industry by-products (whey protein and cardoon leaves), this work supports circular economy principles and promotes sustainability through active packaging solutions that may be applied to different food products.
- Synthesis and Potential Anti-Inflammatory Activity of Floridoside PhosphotriestersPublication . Pinheiro, Luís Pedro Serra; Branco, Paula; Freitas, MarisaFloridoside (2-O-D-glycerol-α-D-galactopyranoside) is a natural product typically found in red algae. It serves as the algae’s carbon reserve, and it is produced as a protective response against osmotic and heat stress. Floridoside has been associated with modulating redox homeostasis and inflammatory responses. These documented activities suggest that structural modifications may influence cellular ox-idative processes. Therefore, our aim was to evaluate whether the newly synthesized families of florido-side phosphotriesters (93a-d and 94a,b) and one acylated floridoside derivative (95) can modulate hu-man neutrophils’ oxidative burst. Synthetic strategies to achieve the desired compounds included gly-cosylation with the thioglycoside donor strategy, having NIS/TfOH as the promoter, and phosphoryla-tion with POCl3 in the presence of pyridine. The compounds were also analyzed for their cytotoxicity with 93a and 94b being cytotoxic at 50 μM while the others showed no cytotoxicity in the tested con-centrations. The detection of the human neutrophils’ oxidative burst was performed using multiple probes [luminol, aminophenyl fluorescein (APF), and amplex red (AR)]. Compound 95, was the most active, demonstrating the ability to prevent the oxidative burst in activated human neutrophils (IC50 = 83 ± 7 μM). All the tested compounds were ineffective in inhibiting APF and AR oxidation. Structure-activity relationships (SAR) suggest that fatty acid chains or the presence of long chains in general are beneficial for activity, which is in accordance with the literature. Results also suggest that the phos-photriester moiety might be redundant for activity or might increase the cytotoxicity of the compounds. These findings highlight the potential of floridoside-based derivatives as valuable scaffolds for targeting key mechanisms in inflammation.
- Tailoring the Properties of Arabinoxylan-Based Films from Corn FiberPublication . Weng, Verónica Lee; Brazinha, Carla; Coelhoso, Isabel; Alves, VítorCorn fiber, an abundant agro-industrial residue from the starch industry, is a rich source of arabinoxylan (Ax), a polysaccharide with inherent film-forming capabilities and prebiotic potential. Films produced directly from arabinoxylan extracts already possess interesting functional properties, including inherent antioxidant activity and a natural UV/visible light barrier. However, for broader application in areas like food packaging, two key characteristics were targeted for improvement: the intense brown color of the extracts, which may not be appealing to consumers, and the high water-sensitivity of the films. This thesis presents a comprehensive strategy to valorize corn fiber by developing arabinoxylan-based films with enhanced aesthetic appeal and water resistance. Initially, arabinoxylan was extracted from corn fiber using an alkaline solution and purified, yielding a polymer primarily composed of xylose (50–52%) and arabinose (37–39%) with an arabinose to xylose (A/X) ratio of 0.72–0.77. During the purification, the smaller compounds (including some phenolic compounds) were partially removed, and this significantly enhanced the extract's antiproliferative effect against the HT29 cell line (EC50 = 0.12 ± 0.02 mg/mL) without cytotoxicity to Caco-2 cells. To create a more neutral-colored film and broaden its consumer appeal, a novel decolorization strategy was developed using the macroporous resin MN102. The adsorption process was systematically optimized, demonstrating high color removal efficiency (initial removal of 63.3%) and excellent reusability of the resin over multiple cycles. The adsorption mechanism was best described by the Freundlich isotherm model, and fixed-bed column breakthrough curves were successfully fitted with the Yan and log-Gompertz models, providing a robust framework for scalable decolorization. To overcome the polymer's inherent hydrophilicity, Ax was chemically modified via acetylation, substituting hydroxyl groups by acetyl groups. This modification, confirmed by infrared analysis (FT-IR), resulted in a polymer and corresponding film with improved thermal properties and enhanced the film's water resistance. Acetylated Ax films plasticized with triacetin exhibited a reduction in water solubility (from 96% to 4%), and a significantly lower water vapor permeability ((0.65±0.07) ×10⁻¹¹ mol·m·m⁻²·s⁻¹·Pa⁻¹) compared to unmodified AX films ((2.89±0.35) ×10⁻¹¹ mol·m·m⁻²·s⁻¹·Pa⁻¹). The acetylation process also resulted in light-colored films (ΔE*ab = 31.09) and maintained their mechanical integrity. In conclusion, this research successfully integrates extraction and purification, resin adsorption, and chemical modification to enhance the functional properties of arabinoxylan. By systematically addressing the challenges of consumer appeal and water sensitivity, this work transforms a low-value by-product into a high-performance biomaterial, yielding light-colored, hydrophobic films with significant potential for sustainable food packaging applications.
- Development of biopolymer films from Opuntia spp. Mucilage for food packaging applicationPublication . Rodrigues, Carolina Pereira; Fernando, Ana Luisa; Coelhoso, Isabel; Souza, VictorThis Ph.D thesis explores the valorization of Opuntia ficus-indica residues, cladodes and fruit peels, as sustainable sources of polysaccharides and natural pigments for the development of bio-based intelligent food packaging. The work combined extraction, material characterization, and functional testing to demonstrate the feasibility of transforming these agro-industrial by-products into value-added biopolymer systems. Initially, polysaccharide-based edible coatings were evaluated on prickly pear fruits. Coatings formulated from pectin, alginate, chitosan, and starch effectively delayed weight loss, maintained firmness and color, and reduced microbial growth during storage, confirming the potential of natural polymers in food preservation. Subsequently, pectin was extracted from Opuntia cladodes using optimized conditions established through response surface methodology. The extracted pectin exhibited a low degree of esterification and high galacturonic acid content. Betalain-rich extracts were obtained as natural colorants with high pH sensitivity. Composite films were developed by blending pectin extracted from cladodes and com- mercial pectin (OFIPC) incorporating montmorillonite (MMT), betalain-rich extracts with or without calcium crosslinking. MMT addition improved mechanical resistance and barrier properties, crosslinking modulates the microstructure and permeability, while betalains conferred colorimetric sensitivity to pH changes. The films demonstrated distinct color transitions in response to ammonia released during salmon spoilage, confirming their functionality as freshness indicators. The films combined strong mechanical performance with low water vapor permeability and rapid chromatic response. exhibited balanced barrier properties, and a color transition from pink to yellow in response to volatile amines generated during fish spoilage. Overall, this work represents a step toward waste valorization and the circular use of plant residues. The research demonstrated that Opuntia ficus-indica is a promising source of biopolymers and pigments for sustainable smart packaging. It highlights the potential of circular approaches that convert agricultural residues into intelligent materials, contributing to reduced plastic dependency and improved food quality monitoring.