Untitled: connecting layers of confusion, sensation and transformation

Dissertação para obtenção do Grau de Mestre em Arte e Ciência do Vidro

Storage of water molecules into biomimetic heterostructures: the role of roughness

The development of devices based on heterostructured thin films of biomolecules conveys a huge contribution on biomedical field. However, to achieve high efficiency of these devices, the storage of water molecules into these heterostructures, in order to maintain the biological molecules hydrated, is mandatory. Such hydrated environment may be achieved with lipids molecules which have the ability to rearrange spontaneously into vesicles creating a stable barrier between two aqueous compartments. Yet it is necessary to find conditions that lead to the immobilization of whole vesicles on the heterostructures. In this work, the conditions that govern the deposition of open and closed liposomes of 1.2-dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (sodium Salt) (DPPG) onto polyelectrolytes cushions prepared by the layer-by-layer (LbL) method were analyzed. Electronic transitions of DPPG molecules as well as absorption coefficients were obtained by vacuum ultraviolet spectroscopy, while the elemental composition of the heterostructures was characterized by x-ray photoelectron spectroscopy (XPS). The presence of water molecules in the films was inferred by XPS and infrared spectroscopy. Quartz crystal microbalance (QCM) data analysis allowed to conclude that, in certain cases, the DPPG adsorbed amount is dependent of the bilayers number already adsorbed. Moreover, the adsorption kinetics curves of both adsorbed amount and surface roughness allowed to determine the kinetics parameters that are related with adsorption processes namely, electrostatic forces, liposomes diffusion and lipids re-organization on surface. Scaling exponents attained from atomic force microscopy images statistical analysis demonstrate that DPPG vesicles adsorption mechanism is ruled by the diffusion Villain model confirming that adsorption is governed by electrostatic forces. The power spectral density treatment enabled a thorough description of the accessible surface of the samples as well as of its inner structural properties. These outcomes proved that surface roughness influences the adsorption of DPPG liposomes onto surfaces covered by a polyelectrolyte layer. Thus, low roughness was shown to induce liposome rupture creating a lipid bilayer while high roughness allows the adsorption of whole liposomes. In addition, the fraction of open liposomes calculated from the normalized maximum adsorbed amounts decreases with the cushion roughness increase, allowing us to conclude that the surface roughness is a crucial variable that governs the adsorption of open or whole liposomes. This conclusion is fundamental for the development of well-designed sensors based on functional biomolecules incorporated in liposomes. Indeed, LbL films composed of polyelectrolytes and liposomes with and without melanin encapsulated were successfully applied to sensors of olive oil.

Biomimetic mineralization: encapsulation in calcium carbonate shells

Calcium carbonate biomineralization is a self-assembly process that has been studied to be applied in the biomedical field to encapsulate biomolecules. Advantages of engineering mineral capsules include improved drug loading efficiencies and protection against external environment. However, common production methods result in heterogeneous capsules and subject biomolecules to heat and vibration which cause irreversible damage. To overcome these issues, a microfluidic device was designed, manufactured and tested in terms of selectivity for water and oil to produce a W/O/W emulsion. During the development of this work there was one critical challenge: the selective functionalization in closed microfluidic channels. Wet chemical oxidation of PDMS with 1M NaOH, confirmed by FTIR, followed by adsorption of polyelectrolytes - PDADMAC/PSS - confirmed by UV-Vis and AFM results, render the surface of PDMS hydrophilic. UV-Vis spectroscopy also confirmed that this modification did not affect PDMS optical properties, making possible to monitor fluids and droplets. More important, with this approach PDMS remains hydrophilic over time. However, due to equipment constrains selectivity in microchannels was not achieved. Therefore, emulsion studies took place with conventional methods. Several systems were tried, with promising results achieved with CaCO3 in-situ precipitation, without the use of polymers or magnesium. This mineral stabilizes oil droplets in water, but not in air due to incomplete capsule formation.

Development of biomimetic microengineered hydrogel fibers for tendon regeneration

Musculoskeletal diseases are one of the leading causes of disability worldwide. Tendon injuries are responsible for substantial morbidity, pain and disability. Tissue engineering strategies aim at translating tendon structure into biomimetic materials. The main goal of the present study is to develop microengineered hydrogel fibers through the combination of microfabrication and chemical interactions between oppositely charged polyelectrolytes. For this, methacrylated hyaluronic acid (MeHA) and chondroitin sulfate (MeCS) were combined with chitosan (CHT). Hydrogel fibers were obtained by injecting polymer solutions (either MeHA or MeHA/MeCS and CHT) in separate microchannels that join at a y-junction, with the materials interacting upon contact at the interface. To evaluate cell behavior, human tendon derived cells (hTDCs) were isolated from tendon surplus samples during orthopedic surgeries and seeded on top of the fibers. hTDCs adhered to the surface of the fibers, remaining viable, and were found to be expressing CD44, the receptor for hyaluronic acid. The synthesis of hydrogel fibers crosslinkable through both physical and chemical mechanisms combined with microfabrication technology allows the development of biomimetic structures with parallel fibers being formed towards the replication of tendon tissue architecture.

Organic-inorganic nanostructured multilayers for calcium phosphate biomineralization

The weak fixation of biomaterials within the bone structure is one of the major reasons of implants failures. Calcium phosphate (CaP) coatings are used in bone tissue engineering to improve implant osseointegration by enhancing cellular adhesion, proliferation and differentiation, leading to a tight and stable junction between implant and host bone. It has also been observed that materials compatible with bone tissue either have a CaP coating or develop such a calcified surface upon implantation. Thus, the development of bioactive coatings becomes essential for further improvement of integration with the surrounding tissue. However, most of current applied CaP coatings methods (e.g. physical vapor deposition), cannot be applied to complex shapes and porous implants, provide poor structural control over the coating and prevent incorporation of bioactive organic compounds (e.g. antibiotics, growth factors) because of the used harsh processing conditions. Layer-by-layer (LbL) is a versatile technology that permits the building-up of multilayered polyelectrolyte films in mild conditions based on the alternate adsorption of cationic and anionic elements that can integrate bioactive compounds. As it is recognized in natureâ s biomineralization process the presence of an organic template to induce mineral deposition, this work investigate a ion based biomimetic method where all the process is based on LbL methodology made of weak natural-origin polyelectrolytes. A nanostructured multilayer component, with 5 or 10 bilayers, was produced initially using chitosan and chondroitin sulphate polyelectrolyte biopolymers, which possess similarities with the extracellular matrix and good biocompatibility. The multilayers are then rinsed with a sequential passing of solutions containing Ca2+ and PO43- ions. The formation of CaP over the polyelectrolyte multilayers was confirmed by QCM-D, SEM and EDX. The outcomes show that 10 polyelectrolyte bilayer condition behaved as a  better site for initiating the formation of CaP as the precipitation occur at earlier stages than in 5 polyelectrolyte bilayers one. This denotes that higher number of bilayers could hold the CaP crystals more efficiently. This work achieved uniform coatings that can be applied to any surface with access to the liquid media in a low-temperature method, which potentiates the manufacture of effective bioactive biomaterials with great potential in orthopedic applications.

Saloplastic membranes as green devices for soft tissue regeneration

Implantable devices must exhibit mechanical properties similar to native tissues to promote appropriate cellular behavior and regeneration. Herein, we report a new membrane manufacture method based on the synthesis of polyelectrolyte complexes (PECs) that exhibit saloplasticity, i.e. variable physical-chemistry using salt as a plasticizer. This is a Green Chemistry approach, as PECs generate structures that are stabilized solely by reversible electrostatic interactions, avoiding the use of harmful crosslinkers completely. Furthermore, natural polyelectrolytes - chitosan and alginate - were used. Upon mixing them, membranes were obtained by drying the PECs at 37ºC, yielding compact PECs without resorting to organicsolvents. The plasticizing effect of salt after synthesis was shown by measuring tensile mechanical properties, which were lower when samples were immersed in high ionic strength solutions.Salt was also used during membrane synthesis in different quan- tities (0 M, 0.15 M and 0.5 M in NaCl) yielding structures with no significant differences in morphology and degradation (around 15% after 3 months in lysozyme). However, swelling was higher (about 10x) when synthesized in the presence of salt. In vitro cell studies using L929 fibroblasts showed that cells adhered and proliferated preferentially in membranes fabricated in the presence of salt (i.e. the membranes with lower tensile strength). Structures with physical-chemical properties controlled with precision open a path to tissue engineering strategies depending on fine tuning mechanical properties and cellular adhesion simply by changing ionic strength during membrane manufacture

Silica nanoparticles as dexamethasone delivery systems able to induce the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

Bioactive glasses, especially silica-based materials, are reported to pres- ent osteoconductive and osteoinductive properties, fundamental char- acteristics in bone regeneration [1,2]. Additionally, dexamethasone (Dex) is one of the bioactive agents able to induce the osteogenic differ- entiation of mesenchymal stem cells by increasing the alkaline phos- phatase activity, and the expression levels of Osteocalcin and Bone Sialoprotein [3]. Herein, we synthesised silica (SiO2) nanoparticles (that present inherent bioactivity and ability to act as a sustained drug delivery system), and coated their surface using poly-L-lysine (PLL) and hyaluronic acid (HA) using the layer-by-layer processing technique. Further on, we studied the influence of these new SiO2-polyelectrolyte coated nanoparticles as Dex sustained delivery systems. The SiO2 nanoparticles were loaded with Dex (SiO2-Dex) and coated with PLL and HA (SiO2-Dex-PLL-HA). Their Dex release profile was evaluated and a more sustained release was obtained with the SiO2-Dex-PLL-HA. All the particles were cultured with human bone marrow-derived mes- enchymal stem cells (hBMSCs) under osteogenic differentiation culture conditions. hBMSCs adhered, proliferated and differentiated towards the osteogenic lineage in the presence of SiO2 (DLS 174nm), SiO2-Dex (DLS 175nm) and SiO2-Dex-PLL-HA (DLS 679nm). The presence of these materials induced the overexpression of osteogenic transcripts, namely of Osteocalcin, Bone Sialoprotein and Runx2. Scanning Elec- tron Microscopy/Electron Dispersive Spectroscopy analysis demon- strated that hBMSCs synthesised calcium phosphates when cultured with SiO2-Dex and SiO2-Dex-PLL-HA nanoparticles. These results indi- cate the potential use of these SiO2-polyelectrolytes coated nanoparti- cles as dexamethasone delivery systems capable of promoting osteogenic differentiation of hBMSCs.

Compact saloplastic membranes of natural polysaccharides for soft tissue engineering

The regeneration of soft biological tissues requires new substitutes that exhibit mechanical properties similar to the native tissue. Herein, thin saloplastic membranes with tunable physical properties are prepared by complexation of chitosan and alginate solutions containing different concentrations of sodium chloride. Polyelectrolyte complexes (PECs) are transferred to flat Petri dishes for compaction into membrane shapes by sedimentation and solvent evaporation. All membranes are resistant to degradation by lysozyme and are stable in solutions with pH values between 1 and 13. Immersing the different membranes in new doping solutions of increasing salt concentrations triggers the typical saloplastic behavior, with a high water absorption and decrease of the rigidity and ultimate tensile strength. The range of such variations is tuned by the sodium chloride amount used in the synthesis: high salt concentrations increase water uptake and tensile moduli, while decreasing the ultimate strength. Cellular assays demonstrate high proliferation rates and viability of L929 fibroblasts seeded onto the most rigid membranes. The results validate the use of saloplastic membranes as soft tissue substitutes for future biomedical applications.

Platelet lysate-based pro-angiogenic nanocoatings

Human platelet lysate (PL) is a cost-effective and human source of autologous multiple and potent pro-angiogenic factors, such as vascular endothelial growth factor A (VEGF A), fibroblast growth factor b (FGF b) and angiopoietin-1. Nanocoatings previously characterized were prepared by layer-by-layer assembling incorporating PL with marine-origin polysaccharides and were shown to activate human umbilical vein endothelial cells (HUVECs). Within 20 h of incubation, the more sulfated coatings induced the HUVECS to the form tube-like structures accompanied by an increased expression of angiogenicassociated genes, such as angiopoietin-1 and VEGF A. This may be a cost-effective approach to modify 2D/3D constructs to instruct angiogenic cells towards the formation of neo-vascularization, driven by multiple and synergistic stimulations from the PL combined with sulfated polysaccharides. Statement of Significance The presence, or fast induction, of a stable and mature vasculature inside 3D constructs is crucial for new tissue formation and its viability. This has been one of the major tissue engineering challenges, limiting the dimensions of efficient tissue constructs. Many approaches based on cells, growth factors, 3D bioprinting and channel incorporation have been proposed. Herein, we explored a versatile technique, layer-by-layer assembling in combination with platelet lysate (PL), that is a cost-effective source of many potent pro-angiogenic proteins and growth factors. Results suggest that the combination of PL with sulfated polyelectrolytes might be used to introduce interfaces onto 2D/3D constructs with potential to induce the formation of cell-based tubular structures.

Development and characterization of lipid-based nanosystems for controlled release of bioactive compounds

Tese de Doutoramento em Engenharia Química e Biológica.

Production of elastic Chitosan/Chondroitin sulphate multilayer films for tissue regeneration

Membrane-like scaffolds are suitable to induce regeneration in many and different anatomic sites, such as periodontal membrane, skin, liver and cardiac tissues. In some circumstances, the films should adapt to geometrical changes of the attached tissues, such as in cardiac or blood vessel tissue engineering applications. In this context, we developed stretchable two-dimensional multilayer constructs through the assembling of two natural-based polyelectrolytes, chitosan (CHT) and chondroitin sulphate (CS), using the layer-by-layer methodology. The morphology, topography and the transparency of the films were evaluated. The in- fluence of genipin, a natural-derived cross-linker agent, was also investigated in the control of the mechanical properties of the CHT/CS films. The water uptake ability can be tailored by changing the cross-linker concentration, which influenced the young modulus and ultimate tensile strength. The maximum extension tends to decrease with the increase of genipin concentration, compromising the elastic properties of CHT/CS films: nevertheless using lower cross-linker contents, the ultimate tensile stress is similar to the films not cross-linked but exhibiting a significant higher modulus. The in vitro biological assays showed better L929 cell adhesion and proliferation when using the crosslinked membranes and confirmed the non-cytotoxicity of the CHT/CS films. The developed free-standing biomimetic multilayer could be designed to fulfill specific therapeutic requirements by tuning properties such as swelling, mechanical and biological performances.

Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins

Addition of a 50 mM mixture of l-arginine and l-glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.

Filmes poliméricos ultrafinos produzidos pela técnica de automontagem: preparação, propriedades e aplicações

The self-assembly technique is a powerful tool to fabricate ultrathin films from organic compounds aiming at technological applications in molecular electronics. This relatively new approach allows molecularly flat films to be obtained on a simple and cheap fashion from various types of material, including polyelectrolytes, conducting polymers, dyes and proteins. The resulting multilayer films may be fabricated according to specific requirements since their structural and physical properties may be controlled at the molecular level. In this review we shall comment upon the evolution of preparation methods for ultrathin films, the process of adsorption and their main properties, as well as some examples of technological applications of layer-by-layer or self-assembled films.

Production of fungal protein by solid substrate fermentation of cactus Cereus peruvianus and Opuntia ficus indica

Agricultural wastes from cactus Cereus peruvianus and Opuntia ficus indica were investigated for protein production by solid substrate fermentation. Firstly, the polyelectrolytes were extracted and used in water cleaning as auxiliary of flocculation and coagulation. The remaining fibrous material and peels were used as substrate for fermentation with Aspergillus niger. Glucoamylase and cellulase were the main enzymes produced. Amino acids were determined by HPLC and protein by Lowry's method. After 120 hours of fermentation the protein increased by 12.8%. Aspartic acid (1.27%), threonine (0.97%), glutamic acid (0.88%), valine (0.70%), serine (0.68%), arginine (0.82%), and phenylalanine (0.51%) were the principal amino acids produced.

Redução de poluentes de efluentes das indústrias de papel e celulose pela floculação/coagulação e degradação fotoquímica

The abatement of recalcitrant lignin macromolecules from effluents of pulp and paper industry was investigated by combined process. Flocculation and coagulation with aluminum sulfate and natural polyelectrolytes extracted from cactus Cereus peruvianus were used in the first step. After separation of solid residues by filtration, the photochemical methods using TiO2 as catalyst were employed for photocatalytic degradation of lignin compounds from solution. The abatement of lignin compounds after flocculation and coagulation was 46%, and after the overall process, the pollutants reduction observed were 66%. The remaining organic compounds may be removed by any biological treatment.