Cancer is one of the leading causes of death worldwide, and current therapeutic options present numerous drawbacks. Chemotherapy, specifically, is associated with widely known side effects. However, and due to the scientific and technologic advances of the past years, a new approach to this dilemma appears in nanomedicine: the use of nanosystems capable of releasing the anticancer drugs in a controlled way and only at the designated cells. These nanosystems may be obtained from numerous materials, including biopolymers, and by various techniques. Specifically, the work described here consists in the synthesis and characterization of nanoparticles obtained by layer-by-layer assembly from amino-modified spherical SiO2 templates (diameter: 234 ± 19 nm). The alternate deposition of biopolymers - alginate (ALG) and chitosan (CH) or lysozyme nanofibrils (LNFs) - on templates pre-loaded with curcumin allowed the creation of nanoparticles covered in ALG/CH (with a diameter of 243 ± 8 nm) or ALG/LNFs (with 242 ± 8 nm). The systematic reversion the zeta-potential and the observation of the particles by scanning electron microscopy (SEM) confirms the deposition of each biopolymeric layer. The present work also includes the evaluation of the release profile of this drug from the multi-layered nanoparticles, demonstrating a more controlled release than with the bare counterparts. The evaluation of their cytotoxic capacity in human liver cancer cell line (HepG2) was posteriorly evaluated through an MTT assay. The cytotoxic effect of layered particles containing CUR was similar to the unlayered counterparts’, demonstrating that the two layers of biopolymers (ALG/CH or ALG/LNFs) do not compromise their cytotoxic potential.

Biomedical research aims to unveil the many secrets of health and disease, since a deeper understanding of the human organism and the illnesses that may affect it is vital for the development of more efficient prevention measures, improved detection strategies, and new treatments. The current standard platform for biomedical testing is the simple bidimensional (2D) cell culture in a monolayer, which has loyally served its purpose in the past decades. However, living organisms are a complex interplay of three-dimensionally (3D) organized tissues, and this organization modifies cell viability, but also their phenotype and differentiation. Considering this, and aiming to find a new steppingstone between the 2D culture models and the in vivo testing in animal models, biomedical researchers have started to explore the creation of 3D living platforms that may be used as closer analogues of the native tissues. Thus, the biological variation of additive manufacturing, known as 3D bioprinting, has emerged as a very relevant technique for the controlled biofabrication of 3D living structures with defined shape and size. 3D bioprinting is defined as the layer-by-layer deposition of “bioinks”, which are formulations composed of living cells, biomaterials and other bioactive components (e.g., growth factors and drugs) to originate living 3D constructs. Hydrogel-based formulations became the most common type of bioinks explored for extrusion-based 3D bioprinting, because of their close resemblance to the native extracellular matrix. Biopolymeric hydrogels have numerous advantages for these endeavors, being biocompatible, biodegradable and easily functionalized. Moreover, biopolymers are often capable of crosslinking under mild conditions (e.g., through temperature, pH or the use of different cations) to create stable living 3D structures. However, certain hydrogels based on biopolymers like polysaccharides and proteins lack the adequate rheological and mechanical properties for these applications. Moreover, biopolymeric hydrogels usually do not possess the functional characteristics of biological tissues (e.g., electrical conductivity) or other functionalities (e.g., drug delivery capabilities) that could be relevant for biomedical applications. Hence, the present thesis aims at developing advanced composite hydrogelbased bioinks by combining different biobased nano- and microstructures with hydrogels based on polysaccharides (viz. carboxymethyl cellulose, alginate and gellan gum), with improved rheological and mechanical properties, but also enhancing their printability, stability and biological performance, while granting them with different functionalities. Herein, hydrogel formulations were loaded with different cell lines (viz. keratinocytes (HaCaT), chondrocytes (ATDC5) and cardiomyocytes (H9c2)) and 3D bioprinted to originate living platforms of distinct types of tissues. Specifically, the first study explores the formulation of a bioink composed exclusively of cellulose (“all-cellulose bioink”), by combining a soluble cellulose derivative, carboxymethyl cellulose (CMC), with nanofibrillated cellulose (NFC) for the 3D bioprinting of both skin (HaCaT) and chondrogenic (ATDC5) cells. The use of different mass proportions of CMC and NFC (viz. 90:10, 80:20 and 70:30) allowed the development of distinct hydrogel-based formulations that showed a good shear-thinning behavior and elastic modulus (G’) recovery rate above 95%, with the addition of NFC increasing their printability from Pr = 0.7 (CMC) to Pr = 0.9 (80:20 and 70:30). The inclusion of NFC also allowed the tailoring of the mechanical properties of the crosslinked hydrogels, reducing their stiffness from 35.92 ± 2.68 MPa (CMC) to 14.14 ± 2.46 MPa (70:30), achieving values within the range of certain biological tissues (viz. cartilage). Simultaneously, the increasing contents of NFC also reduced the degradation rate of the hydrogels in both phosphate-buffered saline (PBS) and culture medium (DMEM) for up to 7 days, from 42.31 ± 2.54% (CMC) to 30.47 ± 0.63% (70:30). The ink that revealed the best combination of properties (viz. 70:30) was loaded with HaCaT or ATDC5 cells and 3D bioprinted, demonstrating the maintenance of high cell viabilities (>80%) within the 3D structures after 7 days post-bioprinting for both cell lines, corroborating the versatility of the formulation and its potential to fabricate different 3D living constructs for biomedical applications. The second study investigated the use of cellulose-based particles (CEp) as carriers of a bioactive compound (curcumin), and their inclusion in an alginate hydrogel to develop drug-releasing bioinks for the 3D bioprinting HaCaT cells. The addition of different contents (1, 5 and 10 wt.%) of the spherical cellulosebased particles (CEpCUR) did not compromise the shear-thinning behavior and the G’ recovery rate of the formulations, increasing their printability from Pr = 0.8 (ALG) to Pr = 0.9 (ALG:CEpCUR_10%). Interestingly, all the resulting crosslinked hydrogels showed a Young’s modulus of around 2.5 MPa, regardless of the CEpCUR contents. However, the presence of CEpCUR particles significantly reduced the degradation rate of the hydrogels in DMEM from 26.3 ± 0.8% (ALG) to 18.7 ± 1.3% (ALG:CEpCUR_10%). Additionally, the structures 3D printed using the ALG:CEpCUR inks were able to successfully release curcumin (with ~70% of cumulative release in PBS after 24 h). After the loading of the formulations with HaCaT cells (1.2 × 106 cells mL–1), the ALG:CEpCUR bioinks were 3D bioprinted, and the obtained living constructs showed good dimensional stability and high cell viability (nearly 90%) after 7 days post-bioprinting, hinting at their potential for instance for wound healing applications. Lastly, the third study developed within the scope of this thesis explored the use of nanostructured hybrids of lysozyme amyloid nanofibrils (LNFs) and gold nanoparticles (AuNPs), “LNF:AuNPs”, as additives in the development of nanocomposite bioinks based on a hydrogel of gellan gum (1.5 wt.%) and carboxymethyl cellulose (1.0 wt.%). The nanocomposite ink formulations revealed a shear-thinning behavior and high G’ recovery rate (>85%), and an electrical conductivity similar to the cardiac tissue (~3.0 mS cm-1). The inclusion of LNF:AuNPs decreased the degradation rate of the crosslinked hydrogels from above 30% to 13.8 ± 2.4%, while improving their antioxidant activity (from 22.0 ± 0.8% to 75.1 ± 0.3%). Moreover, the crosslinked hydrogels revealed a Young’s modulus within the range of the myocardium (<100 kPa) and a highly porous morphology (>70%) that is beneficial for biomedical applications. The 3D bioprinting of the best performing formulation with H9c2 cells is able to originate living structures where cells maintain high cell viability values (around 90%) for up to 7 days, confirming the biocompatibility of the bioinks and their potential for the biofabrication of 3D structures that may find application as cardiac tissue platforms. In sum, the composite bioinks developed within the framework of this thesis and described herein are an important addition to the world of biopolymeric hydrogel-based bioinks for 3D bioprinting, presenting new approaches that may inspire further innovation and open new doors in this realm. Moreover, these studies highlight the enormous potential of using different micro- and nanostructured additives as modifiers of hydrogel-based bioinks, originating formulations with excellent features and diverse functionalities for the 3D bioprinting of distinct cell lines.

This integrative literature review study aimed to analyze the clinical and laboratory findings, in addition to the treatment of case reports of CADASIL from the last 5 years. The database used in the search was PubMed, with the following descriptors: “CADASIL”, “case report”, in addition to the use of the Boolean operator (AND). The final sample was 25 baseline studies. Although CADASIL is caused by a genetic mutation, there was a case in which COVID-19 infection was the beginner of the manifestations of a patient with a previous mutation. In the exams, most have shown damages in MRI, with confluent hypertensive foci, microinfarcts, stenosis and even brain atrophy. Cases were also recorded in which the disease presented parkinsonian syndrome, symptoms of atypical paraplegia, mood alterations and transient ischemic attack. In the study it was perceptive the manifestations in families with this genetic disease. The symptons can be manifested in hetero or homozygous, and may cause mental disorders, headache and stroke. The clinical variety shows its complexity, requiring studies to understand all its pathophysiology.There is no cure for this disease, so its treatments were mainly to control the symptoms.