The innate negative charge of glycosaminoglycans (GAG) is in the origin of their bioactivity: it drives their spontaneous complexation with positively charged biomolecules and ions, regulating homeostatic functions by protein stabilization, protection and activation. Copycatting these interactions enables different supramolecular approaches towards the assembly of biofunctional devices. Such approaches allow processing under physiological conditions and thus, they are of unprecedented biocompliance for device build-up compared to conventional methods based on chemical cross-linkers, volatile and organic solvents, and high temperatures. We review different set-ups based on GAG electrostatic complexation and showcase their application towards the development of diverse therapeutic systems. We also discuss challenges associated with GAG complexation into intricate three-dimensional networks holding back the widespread use of these methods. Finally, we anticipate that conscious choice of GAG with distinct polyanionic strength and specific bioactivity will make possible the fabrication of constructs with personalized and customized properties in the nearest future.

Este relatório detalha o desenvolvimento de uma bancada de testes em válvulas, estruturada em dois módulos principais: o módulo de controlo e o módulo de suporte e fixação. A necessidade deste projeto emergiu da análise crítica das instalações de certificação existentes na empresa, que revelou tanto as vantagens quanto as limitações do sistema em uso, fornecendo diretrizes claras para a possível reutilização, ou não, de componentes. O primeiro capítulo do relatório concentra-se na avaliação das configurações atuais, descrevendo os componentes individuais e suas funcionalidades, e avaliando o que poderia ser adaptado para o novo projeto. O segundo capítulo apresenta um levantamento do estado da arte, explorando desenvolvimentos recentes em bancadas de controlo, tecnologias associadas às válvulas e um estudo sobre prensas hidráulicas, fundamentando assim a abordagem adotada no projeto. A fase de projeto conceptual foi abordada com uma distinção clara entre os dois módulos. Para o módulo de controlo, foram concebidos diferentes designs, visando acomodar as funções essenciais identificadas, como a realização dos testes nas válvulas, o controlo da percentagem de abertura e o monitoramento do nível de líquido refrigerante no tanque. A cada função foi atribuída uma solução conceptual específica que se interligavam no conceito da bancada de controlo. Já o módulo de suporte e fixação foi projetado com o objetivo de implementar tecnologia hidráulica e disposição horizontal, com vários conceitos sendo explorados até se chegar a uma solução que atendesse tanto às expectativas da empresa quanto aos requisitos técnicos do projeto. Na fase de projeto detalhado, todas as soluções conceptuais foram revistas e refinadas, culminando na seleção e dimensionamento de componentes para cada subsistema e adaptação e alterações para uma melhor integração das sub soluções. A fase de desenvolvimento baseou-se na construção de protótipos: um protótipo completo replicando o projeto detalhado para o módulo de suporte e fixação, equipado com tecnologia hidráulica e configurado horizontalmente, e um protótipo reduzido para o módulo de controlo, que introduziu a realização de testes preliminares de sensores e de leitura de variáveis críticas. Este relatório conclui que o projeto não só atende às necessidades atuais da empresa, como também estabelece uma fundação sólida para futuras expansões e melhorias. A execução bem-sucedida do módulo de suporte e fixação demonstra a viabilidade da disposição horizontal e da tecnologia hidráulica, prometendo aumentar a eficiência e a precisão dos testes de válvulas. As limitações no desenvolvimento do módulo de controlo devem-se à prioridade da empresa em termos de testes do projeto sugerindo que futuras investigações deverão concentrar-se na integração completa da bancada projetada.

The modification of surfaces has been a key aspect in biology and biotechnology, for applications including cell expansion, biomaterials development and preparation of substrates for regenerative medicine. In this thesis, the layer-by-layer (LbL) technique was employed in the modification of surfaces for multiple purposes, namely for films with improved adhesiveness, enhanced cell adhesion, drug delivery capsules, and magnetic spatial positioning. The working hypothesis was that LbL could be used in the modification of surfaces not only planar but also three-dimensional, using solely polymeric materials from a variety of natural origins or obtained by recombinant routes. Herein, chitosan (CHI), a well-known polycation with marine origins, was used recurrently as an ingredient in various multilayer formulations, driven by intermolecular forces such as electrostatic and hydrophobic interactions. First, multilayer coatings of CHI and an adhesive bacterial exopolysaccharide, levan, were fabricated. As a control, CHI and alginate films – two marine polysaccharides often regarded as good natural adhesives – were assembled in parallel. The adhesive strength of 50 CHI/levan bilayers was determined to be about 3 times higher than the control. The adhesion of L929 cells was also higher in levan-based films. Next, CHI was used along polyanionic elastin-like recombinamers (ELRs), a recombinant class of elastin-like polymers, exhibiting the cell adhesion motif arginine-glycine-aspartic acid (RGD). Although electrostatic interactions were relied upon to construct LbL coatings, they were not the sole mechanism of buildup between CHI and ELRs. The film construction of CHI and one of nine ELRs differing in amino acid content, length and biofunctionality was followed in situ at pH 4.0 and 5.5 using a quartz-crystal microbalance. Their thicknesses were estimated using the Voigt-based model for viscoelastic films, revealing that thicker films were obtained in the presence of hydrophobic interactions between ELRs and partially neutralized chitosan, i.e., when the pH was adjusted to 5.5, near its pKa. ELR/alginate coatings were also monitored, completing a total of 36 combinations studied. The results agreed with the ones from the CHI-based films: thicker films were obtained for partially neutralized alginate, at a pH value of 4.0. Bidimensional coatings of CHI and CHI/ELR-RGD were demonstrated to be stimuli-responsive by means of wettability measurements: they exhibited a moderate hydrophobic surface but switched to an extremely hydrophilic one when temperature, pH or ionic strength were raised above 50ºC, 11, or 1.25 M, respectively. The presence of RGD enhanced the adhesion of SaOs-2 cells, in comparison to films ending either in CHI or a nonbioactive RDG analogue. CHI/ELR-RGD coatings were extrapolated to the third dimension as spherical microcapsules assembled around calcium carbonate particles. Aiming at drug administration, two mechanisms were studied: (i) simple diffusion out of the microcapsules, and (ii) cellular uptake. In the first case, release studies at 25 and 37°C with “bovine serum albumin”-loaded microcapsules demonstrated that they provided a sustained release over 14 days, but with reduced capsule permeability at physiological temperature, due to the temperature-induced molecular transition of ELRs. Furthermore, a higher numbers of layers provided an added barrier to the diffusion of the encapsulated protein. The microcapsules were also noncytotoxic towards L929 cells. In the second case, the internalization efficacy and intracellular traffic of RGD- or nonbioactive RDG-functionalized microcapsules by human mesenchymal stem cells (hMSCs) was assessed. The data suggests that microcapsules were internalized mainly through macropinocytosis and that surface functionalization did not play a significant role on this phenomenon, although intracellular processing was faster for RGD-functionalized microcapsules. Both microcapsule types were noncytotoxic toward hMSCs for microcapsule/cell ratios between 5:1 and 100:1. Finally, inspired by the complex hierarchical and compartmentalized structure of cells, liquefied alginate macroscopic spheres coated with a chitosan/alginate shell were conceived. Within this liquefied environment, rhodamine and multilayer microcapsules were confined, with the latter encapsulating further either rhodamine or magnetic nanoparticles (MNPs). At 25 and 37ºC, rhodamine when encapsulated within the inner microcapsules showed a sustained release profile, with the diffusion kinetics being even more reduced at 25ºC. The release of rhodamine encapsulated in the outer liquefied alginate compartment did not show significant differences as a function of temperature. Encapsulating MNPs within the microcapsules provided magnetic responsiveness to the whole compartmentalized device and guided mobility capability. The developed work shows that LbL is a versatile technique that provides the means to develop a wide array of systems useful in biomedical applications, from adhesive and cell seeding planar supports to three dimensional structures for smart drug delivery, guided therapies and customized microreactors as disease models and microtissue production in laboratory.

Extensive research has been conducted on biomimetic interfaces mimicking the complex and diverse microenvironment of cell membranes to gain insights into bioactive compound interactions and membrane biophysics modulation. The present study proposes an innovative approach that combines five prospective label-free methodologies (derivative spectroscopy, synchrotron small- and wide-angle X-Ray scattering, attenuated total reflectionâ Fourier-transform infrared spectroscopy, quartz-crystal microbalance with dissipation, and surface plasmon resonance) to showcase their synergistic capabilities and complementarity in investigating drugâ membrane interactions. This multitechnique approach combines the real-time monitoring of the adsorption process under continuous flow conditions with the steady-state perspective of this process. As a proof of concept, the interaction of three bioactive compounds (caffeine, testosterone, and diclofenac) with two biomimetic membrane interfaces (multistacked lipid bilayers and supported lipid bilayers) mimicking the more ordered lipid transient phases, with and without cholesterol (lo and so), that are responsible for a variety of membrane-associated biological activities, is investigated. The biophysical effects of the bioactives are discussed using complementary data from real-time and steady-state experiments, including membrane adsorption and distribution, predicted location, and induced changes in order and fluidity, encompassing bilayer thickness, hydration, and area per lipid molecule.

We describe the assembly of layer-by-layer (LbL) ultrathin films containing bioactive hydroxyapatite (HAp) rod-shaped nanoparticles with mineralizing capacity. Monodisperse 96 nm long and 9 nm wide HAp nanorods with a surface charge of â 14 mV were produced with a microfluidic system. The negatively charged HAp nanorods were assembled with the polycation poly-L-lysine (PLL) in LbL fashion. The successful deposition of alternating layers was confirmed by quartz-crystal microbalance with dissipation monitoring. The Voigt-based viscoelastic model demonstrated steady film growth where three PLL/HAp bilayers reached a thickness of 70 nm. The bioactivity of [PLL/HAp]3 was evaluated in vitro by following the formation of a mineralized hydroxyapatite layer in simulated body fluid (SBF). X-ray diffraction, energy-dispersive X-ray spectroscopy and scanning electron microscopy (SEM) demonstrated formation of a crystalline hydroxyapatite layer and complete surface coverage within 7 days. SaOs-2 osteoblasts-like cells attached to the mineralized surfaces and developed longer filopodia extensions when compared to non-mineralized samples. Our results showed that [PLL/HAp]3 films are feasible osteoconductive coatings applicable to orthopedic implants and fixation devices.

Breast cancer is resistant to conventional treatments due to the specific tumour microenvironment, the associated acidic pH and the overexpression of receptors that enhance cells tumorigenicity. Herein, we optimized the synthesis of acidic resorbable calcium carbonate (CaCO3) nanoparticles and the encapsulation of a low molecular weight model molecule (Rhodamine). The addition of ethylene glycol during the synthetic process resulted in a particle size decrease: we obtained homogeneous CaCO3 particles with an average size of 564 nm. Their negative charge enabled the assembly of layer-by-layer (LbL) coatings with surface-exposed hyaluronic acid (HA), a ligand of tumour-associated receptor CD44. The coating decreased Rhodamine release by two-fold compared to uncoated nanoparticles. We demonstrated the effect of nanoparticles on two breast cancer cell lines with different aggressiveness â SK-BR-3 and the more aggressive MDA-MB-231 â and compared them with the normal breast cell line MCF10A. CaCO3 nanoparticles (coated and uncoated) significantly decreased the metabolic activity of the breast cancer cells. The interactions between LbL-coated nanoparticles and cells depended on HA expression on the cell surface: more particles were observed on the surface of MDA-MB-231 cells, which had the thickest endogenous HA coating. We concluded that CaCO3 nanoparticles are potential candidates to carry low molecular weight chemotherapeutics and deliver them to aggressive breast cancer sites with an HA-abundant pericellular matrix. 

Nervous system disorders are characterized by a progressive loss of function and structure of neurons that ultimately leads to a decline in cognitive and motor functions. In this study, we used interfacial polyelectrolyte complexation (IPC) to produce fibers for neural tissue regeneration. IPC is a processing method that allows spinning of sensitive biopolymers. The rate of spinning and the properties of the used biopolymers (charge and molecular weight) influence different characteristics of the fibers such as size and stability, among others. We used two major components of the neuronal stem cell niche, the polycationic collagen (Col) and the polyanionic hyaluronic acid (HA), to obtain bioactive fibers. We tested HA with different molecular weights and found that HA with medium and high molecular weights (350 and 1200 kDa, respectively) enabled drawing of microfibers with a homogeneous distribution of Col and HA, whereas low-molecular-weight HA (40 kDa) did not allow spinning. The obtained microfibers showed high swelling ability in a physiological buffer: their diameters increased more than 5-fold from their dry state. At these conditions, the tensile storage moduli of the fibers were similar to nervous tissues. Collagenase and hyaluronidase did not change the morphology of the fibers for up to 3 days but reduced their moduli 2- to 3-fold. Assays with PC12 neuronal-like cells showed that IPC microfibers support cell adhesion and viability regardless of the molecular weight of the used HA.

In the last few years, several strategies have been proposed to fabricate scaffolds for tissue engineering (TE) applications; however, they are based on harsh and timeâ consuming techniques. The choice for natural polymers such as cashew gum (CG) allows to circumvent the demands of biocompatibility and degradability of TE systems. In this work, CG, a polysaccharide derived from Anacardium occidentale trees, is functionalized with aldehyde groups through periodate oxidation. The resultant oxidized cashew gum (CGO) is mixed with gelatin (GE) to yield a covalently crosslinked hydrogel. CGO/GE sponges are obtained by employing a freezeâ drying methodology to the previously obtained hydrogels. The mechanical properties, swelling ability, and porosity of the GE/CGO sponges are tuned by using CGO with different degrees of oxidation. The resultant sponges can maintain high levels of water absorption and recover their initial mechanical properties after cyclic compression. Moreover, these porous and mechanically robust devices can support the adhesion and proliferation of cells, which can envision their application for the regeneration of soft tissues.