In the area of tissue engineering different approaches have been studied, so far, for promoting regeneration or replacement of damaged tissues. Among the different materials developed, hydrogels, due to their biocompatibility and similarities with the native extracellular matrix, have emerged as suitable candidates for being used for different therapeutic purposes. Herein, photocrosslinkable hydrogels, composed by chitosan methacrylamide (ChMA) and gelatin methacrylamide (GelMA) were crosslinked by ultraviolet (UV) light, using Irgacue 2959 as photoinitiator. The morphological, physicochemical and biological properties of the hydrogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and nuclear magnetic resonance. The obtained results demonstrated that the developed hydrogels possess suitable properties for being used as 3D constructs on several areas of tissue engineering. Furthermore, these properties may allow their future application as space filling agents or as delivery vehicles of bioactive molecules and cells.

Cardiovascular disease is the leading cause of morbidity and mortality among industrialized countries. Vascular grafts are often required for the surgical treatments. Considering the limitations associated with the use of autografts and with the currently available synthetic materials, a growing demand in tissue engineered vascular grafts has been registered. During the work here described, electrospinning technique was used to prepared fibrous matrices to be applied as vascular implants. For that purpose, electrospun polycaprolactone (PCL) fibrous mats were produced and afterwards coated with different hydrogel formulations based in photocrosslinkable gelatin (GelMA) and the macromers poly(ethylene glycol) acrylate (PEGA) and poly(ethylene glycol) diacrylate (PEGDA). These were further photocrosslinked under UV irradiation using Irgacure® 2959 (by BASF) as the photoinitiator. The suitability of the coated scaffolds for the intended application, was evaluated by assessing their chemical/physical properties as well as their interaction with blood and endothelial cells.

Gold core silica shell (AuMSS) nanorods present excellent physicochemical proper-ties that allow their application as photothermal and drug delivery agents. Herein,AuMSS nanorods were dual‐functionalized with Polyethylene glycol methyl ether(PEG‐CH3) and Gelatin (GEL) to enhance both the colloidal stability and uptake byHeLa cancer cells. Additionally, the AuMSS nanorods were combined for the firsttime with IR780 (a heptamethine cyanine molecule) and its photothermal and pho-todynamic capacities were determined. The obtained results reveal that the en-capsulation of IR780 (65 μg per AuMSS mg) increases the photothermal conversionefficiency of AuMSS nanorods by 10%, and this enhanced heat generation wasmaintained even after three irradiation cycles with a NIR (808 nm) laser. Moreover,the IR780‐loaded AuMSS/T‐PEG‐CH3/T‐GEL presented≈2‐times higher uptake inHeLa cells, when compared to the non‐coated counterparts, and successfullymediated the light‐triggered generation of reactive oxygen species. Overall, thecombination of photodynamic and photothermal therapy mediated by IR780‐loadedAuMSS/T‐PEG‐CH3/T‐GEL nanorods effectively promoted the ablation of HeLacancer cells.

Hydrogels and lyophilisates were obtained by chemical crosslinking of gelatin using N-hydroxysuccinimide and N, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The systems were characterized with respect to the degree of crosslinking, morphology, water uptake, in vitro drug release and biocompatibility studies. Pilocarpine hydrochloride, a drug for the treatment of glaucoma, was loaded by soaking in an aqueous solution containing the drug. In vitro, the released drug percentage varied between 29.2% and 99.2% in 8 h of study. The release data were fitted to the Korsmeyer–Peppas equation to calculate the release exponent, which indicated anomalous transport for the release of pilocarpine. The corneal endothelial cell culture tests indicated that the prepared biomaterials are not cytotoxic.

Mitochondrial gene therapy seems to be a valuable and promising strategy to treat mitochondrial disorders. The use of a therapeutic vector based on mitochondrial DNA, along with its affinity to the site of mitochondria, can be considered a powerful tool in the reestablishment of normal mitochondrial function. In line with this and for the first time, we successfully cloned the mitochondrial gene ND1 that was stably maintained in multicopy pCAG-GFP plasmid, which is used to transform E. coli. This mitochondrial-gene-based plasmid was encapsulated into nanoparticles. Furthermore, the functionalization of nanoparticles with polymers, such as cellulose or gelatin, enhances their overall properties and performance for gene therapy. The fluorescence arising from rhodamine nanoparticles in mitochondria and a fluorescence microscopy study show pCAG-GFP-ND1-based nanoparticles' cell internalization and mitochondria targeting. The quantification of GFP expression strongly supports this finding. This work highlights the viability of gene therapy based on mitochondrial DNA instigating further in vitro research and clinical translation.

Fining wines are used to ensure the physicochemical stability and prevent the formation of hazes and deposits. Several fining agents (gelatin, isinglass, bentonite, etc.) are used by winemakers. These products are animal proteins or of mineral origin. However, some strict vegetarians do not accept any beverage treated with products of animal origin. Consumers requirements have given origin to new alternatives to these products, such as proteins of plant origin and yeast extracts. This work aimed to evaluate the effect of four fining agents, two vegetable proteins [pea protein and patatin, a protein extract from potatoes (Solanum tuberosum)], one yeast extract and one gelatin, on phenolic composition, volatile composition and sensory profile. The fining agents were used at the minimum (D1) and maximum dosages (D2) suggested by the respective technical sheet. Unfined wine was used as a control (D0). The red wine 2015, was produced in the Escola Superior Agrária de Santarém winery, in Tejo region. The wine is a blend of Tinta Roriz, Syrah and Alicante Bouschet. Total phenolic Index (IPT), total anthocyanins (Ant) and total tannins (Tan), colour intensity (CI) and hue were determined according the International Organization of Vine and Wine (OIV). The turbidity was also evaluated. The analysis of the volatile compounds was done by GC-MS and the sensorial analysis was performed by five expert panellists, members of the ‘Comissão Vitivinícola Regional of Tejo (CVRTejo)’. The attributes of the wine, corresponding to the visual (limpidity and colour), nose (typicity, intensity and quality) and taste senses (typicity, intensity, persistence and quality, as well as the harmony (overall judgment). According the results obtained, the vegetable proteins and the yeast extract used did not influence the wine phenolic composition: IPT (60,04±2,735), Ant (443,96±22,32 mg/L), Tan (3034,33±189,89 mg/L), CI (10,54±0,47), Hue (0,711±0,017). However, the tannin content decreased in fining wines (untreated wine 3178,47±36,24 mg/L; D1: 2980,59±238,73 mg/L; D2 2943,96±154,27 mg/L). The turbidity decreased significantly in fining wines (untreated wine 2,53±0,00; D1 1,68±0,61; D2 1,12±0,47); the gelatin was the less efficient in the decrease of turbidity (2,10±0,46). The results indicate that the fining agents did not significantly remove isobutyl alcohol, isoamyl alcohols, acetoin, ethyl lactate, diethyl succinate, 2,3-butanediol-meso and 2,3-butanediol-levo, 1-hexanol, - butyrolactone, octanoic acid, 2-metil-propanoic acid, diethyl succinate, 2-phenylethanol and ethyl vanilate. As with regards to the hexanoic acid, the ethyl 3-hydroxybutyrate and the ethyl 3-methylbutyl succinate, these decreased when the treatment was carried out with patatin, pea protein or yeast extract. These results are in agreement with those obtained in the sensory analysis, in which the tasters did not detect significant differences between the wines treated with different fining agents.

Soft liquified capsules are explored for various biotechnological applications owing to their versatility and protective nature. However, it is challenging to assess and control their internal environment post-production without compromising their structural integrity. This study explores liquid capsules with shells created from gelatin modified with hydroxypyridinone groups and coordinated with iron ions to enable access to and control over their internal content. Using glycerol as a cryoprotectant prevents ice crystal formation in gelatin-derived hydrogel pores during storage at -20ºC. The hygroscopic properties provided by glycerol effectively preserve the structural and self-healing features of the shell over time, supporting large-scale production of off-the-shelf containers. As a proof-of-concept, we show the ability to manipulate the internal content and real-time analysis of internal pH, oxygen and protein levels. The nature of these capsules allows them to closely emulate the elasticity and self-healing of natural cell membranes, enabling in situ modulation of the internal content without compromising the capsule structural integrity. These findings support the development of universal incubator units for in vitro studies, advancing bioreactors, sensors, and frameworks crucial for bioengineering microtissues across diverse applications.

The growing demand for three-dimensional (3D) platforms that successfully mimic the cellular microenvironment present in native human tissues and allow reducing animal testing in the early stages of biomedical research, like drug screening and delivery, disease research, and tissue engineering and/or transplantation, is leading to the search of novel fabrication strategies of 3D living tissue analogues. Thus, 3D bioprinting, the biological branch of 3D printing, has emerged as an excellent option to produce these 3D living structures. This technology consists of the deposition of bioinks, composed of biomaterials, living cells, and biological components (e.g., growth factors), in a layer-by-layer fashion and following spatial patterns using computer-aided design (CAD) to form 3D living tissue analogues. In this vein, hydrogels are the most used class of bioinks due to their crosslinked polymeric networks that resemble the cellular microenvironments. Hydrogels are easily prepared by the crosslinking of synthetic or natural polymers, yet natural polymers have many advantages, including biocompatibility, ease of functionalization, and biodegradability in physiological conditions with the formation of nontoxic byproducts. Nonetheless, the natural-based hydrogels usually have poor rheological and mechanical properties. Hence, the present Thesis aims at engineering advanced nanostructured hydrogel-based bioinks with superior rheological and mechanical features, printability, stability, and cell viability, through the combination of nanofibrillated cellulose (NFC), a biobased class of nanofibers with unique features, with other biopolymers, namely gelatin (Gel), gellan gum (GG) and FucoPol (FP) for the extrusion-based bioprinting of different cell lines, specifically hepatocarcinoma cells (HepG2), human keratinocytes (HaCaT) and melanoma cells (A375). The first study focused on the formulation of NFC-reinforced Gel-hydrogel-based bioinks produced following a simple approach using genipin as the crosslinking agent, avoiding the need of previous chemical modifications of either the Gel or NFC. The developed bioinks were exploited to bioprint HepG2 cells. First, Gel/NFC inks with different mass compositions, namely 90:10, 80:20, 70:30, and 60:40, were prepared and characterized. The addition of NFC to the Gel-based inks contributed to their shear-thinning behavior and the increase of the Young's modulus (from 4.7 ± 0.9 MPa to 12.1 ± 1.1 MPa for the inks 90:10 and 60:40) and the compressive stress at 80% (from 0.5 ± 0.1 MPa to 1.5 ± 0.2 MPa) of the corresponding crosslinked Gel/NFC hydrogels. The degradation rate of the crosslinked hydrogels decreased from 22.52 ± 1.88% (90:10) to 6.95 ± 0.11% (60:40) after 72 h of incubation in Dulbecco's Modified Eagle's Medium (DMEM) and from 100% (90:10) to 15.15 ± 2.26% (ink 60:40) in phosphate buffer saline (PBS). The printability of the inks and the shape fidelity of the obtained constructs were also enhanced by the addition of NFC (Pr≈0,9), allowing the printing of more intricate structures. The crosslinked Gel/NFC hydrogels were non-cytotoxic towards HepG2 cells, with cell viabilities always above 85%. The ink with the best combination of properties (70:30) was laden with the chosen HepG2 cells (2 × 106 cells mL-1) and bioprinted, which translated into elevated cell viabilities (90 ± 4%) up until 14 days after bioprinting. Additionally, it was confirmed that HepG2 cells maintained their metabolic activity, which proved the enormous potential of using Gel/NFC bioinks for the bioprinting of this cell line while providing an adequate environment for cells to thrive towards the creation, for example, of a hepatocarcinoma tumor model for novel drugs/therapeutics testing. The second study investigated the formulation of a novel bioink composed of NFC and GG in different mass proportions (NFC/GG), viz. 90:10, 80:20, 70:30, and 60:40, for the bioprinting of HaCaT cells. The innovative combination of NFC with GG translated into an improvement of the rheological and storage (G’) modulus recovery of the inks, specifically from 70.05 ± 3.06% (90:10) to 82.63 ± 1.21% (60:40). The mechanical properties of the resulting NFC/GG hydrogel inks, particularly the Young’s modulus and compressive stress at 30%, were, also enhanced, specifically from 114.0 2 ± 10.93 kPa (90:10) to 337.16 ± 34.03 kPa (60:40) and from 18.27 ± 1.32 kPa (90:10) to 47.17 ± 3.59 kPa (60:40), respectively. The degradation rates of the corresponding crosslinked hydrogels decreased from 7.58 ± 1.37% (90:10) to 2.48 ± 0.21% (60:40) after 7 days of incubation in DMEM and from 9.35 ± 3.18% (90:10) to 3.96 ± 1.72% (60:40) in PBS. The developed inks showed good printability with Pr values near 0.9 for all inks. Additionally, they were non- cytotoxic towards the HaCaT cell line, with cell viabilities consistently above the 70% threshold for up to 72 h. The ink with the most promising characteristics (60:40) was loaded with HaCaT cells (3 × 106 cells mL-1) and bioprinted. Cell viability remained high (90 ± 3%) up until 7 days post-bioprinting. The obtained data revealed that the combination of the two polysaccharides (NFC and GG) allowed the fabrication of a hydrogel-based bioink with superior properties that could be used for 3D bioprinting of skin cells, for example, for the fabrication of patches for wound healing. Lastly, and following the same premise, a novel bioink was developed through the combination of NFC with a recently discovered fucose-rich polysaccharide, FP, for the bioprinting of A375 and HaCaT cell lines. The inks with different NFC/FP mass proportions, viz. 1:1, 2:1, 3:1 and 4:1, were prepared, originating the following formulations NFC1FP, NFC2FP, NFC3FP and NFC4FP. An ink composed solely of FP (NFC0FP) was also prepared for comparison purposes. Once again, NFC improved the rheological features of the resulting inks perceived by the increase in the shear viscosity from 1.39 ± 0.03 Pa∙s (NFC0FP) to 2933.7 ± 137.9 Pa∙s (NFC4FP). The mechanical properties were also improved by the addition of NFC, specifically Young’s modulus from 0.12 ± 0.04 MPa (NFC0FP) to 2.45 ± 0.05 MPa (NFC4FP) and the compressive stress at 80% strain from 0.014 ± 0.003 MPa (NFC0FP) to 0.13 ± 0.01 MPa (NFC4FP). The presence of NFC contributed to a notorious decrease in the degradation rate in different media. Specifically, from 16.77 ± 3.61% (NFC2FP) to 10.61 ± 3.61% (NFC4FP), in DMEM, and from 21.82 ± 1.28% (NFC2FP) to 14.67 ± 1.71% (NFC4FP), in PBS. It is worth mentioning that the inks with lower NFC content, namely NFC0FP and NFC1FP, disintegrate completely after 6 h and 8 h, respectively, in both media. These improved features resulted in higher printing fidelity with Pr values around 0.9 for the inks NFC2FP, NFC3FP, and NFC4FP. Moreover, the NFC/FP hydrogels were non-harmful towards both cell lines up to 72 h since the cell viabilities were always above 88%. Bioprinting A375-cell laden (2 × 106 cells mL-1) and HaCaT-cell laden (3 × 106 cells mL-1) NFC3FP bioinks, resulted in high cell viabilities (always above 88%) throughout 21 days post-bioprinting. With this work, a new application was unveiled for FP- based hydrogels with the possibility of bioprinting melanoma models with high cell viability for novel drug/therapeutics testing (A375-laden bioink) or functional living wound healing dressings (HaCaT-laden bioink). In sum, all developed nanostructured hydrogel bioinks represent an important contribution to the growing realm of biopolymeric hydrogel bioinks while highlighting the potential of using NFC as a reinforcing agent for creating bioinks with superior features for 3D bioprinting of different cell lines.

Laccases are applicable in numerous reactions as part of industrial significance, namely in polymer industry, bioremediation, biomass conversion and wine industry. However, industrial applications of laccases are limited due to their poor recyclability and difficulty to isolate from the reaction system, which results in high commercial costs. The main considerations of enzyme immobilization are compatibility, activity, and reusability. Thus, magnetic hybrid nanoparticles have been considered as one of promising materials for laccase immobilization support. Magnetic nanoparticles (MNPs) core afford facile magnetic separation process. Hybrid organic-inorganic shell creates mechanically tuneable material and provide hydroxyl and amino groups, enabling high-affinity interactions with enzymes. In this work, magnetic hybrid NPs modified with silica-gelatin-alkoxysilane were prepared employing versatile synthetic route and characterized by FTIR spectroscopy, elemental, TEM, and zeta potential analysis. Fe₃O₄@SiO₂/Gel-A/ICPTES exhibited the highest activity compared to other magnetic hybrid NPs, thus it was selected as a support material for laccase. Considering the recovered activity and recyclability, laccase immobilization on Fe₃O₄@SiO₂/Gel-A/ICPTES at pH 5 with 3 h of contact time using higher concentration of laccase was considered as the most feasible immobilization condition. The Michaelis-Menten kinetic parameters of laccase immobilized at the optimum conditions showed a Vₘₐₓ of 1.25 μmol.gˉ¹.minˉ¹ and a KM of 75.83 μM, which was approximately four times higher than free laccase. Immobilized laccase displayed a fair recyclability, with 54% of activities retained in the 5th cycle. However, the immobilization of laccase on Fe₃O₄@SiO₂/Gel-A/ICPTES generally displayed low immobilization yields (<20%). Therefore, further adjustments such as enzyme-to-material proportion and immobilization temperature are necessary.

We report a versatile synthetic route for the preparation of nearly monodispersed spherical gelatin-silica hybrid nanoparticles. These hybrids have been synthesized by means of a non-emulsion sol–gel method involving the base-catalyzed hydrolysis and condensation of gelatin modified with different functional coupling agents. The method is effective for preparing hybrids of both gelatin type A and B, and the alkoxysilane coupling agents 3-isocyanatopropyltriethoxysilane (ICPTES) and 3-glycidoxypropyltrimethoxysilane (GPTMS). The ensuing gelatin-silica hybrid nanoparticles are spherical and uniform in morphology, with average sizes between 100 and 120 nm. The ICPTES seems to be more adequate to produce uniform nanospheres with narrow size distribution regardless the type of gelatin, while using GPTMS increases the roughness of the particles’ surface. The hybrids are thermo-sensitive in the temperature range 25–70 °C, undergoing reversible volume transitions in response to thermal stimuli, which is of great relevance for biomedical applications such as thermally controlled drug-delivery systems. The synthetic approach was successfully employed to coat magnetic iron oxide nanoparticles with thin shells of a few nanometers of hybrid composition to obtain hybrid nanoparticles with magnetic features.