Stimuli-responsive systems based on elastin-like recombinamers for biomedical applications

Los recombinámeros de tipo elastina (ELRs) se basan en la repetición de ciertos pentapéptidos que están presentes en la elastina natural y que se pueden producir con un extraordinario grado de complejidad y control mediante técnicas de ingeniería genética. El trabajo compilado aquí es parte de un proyecto a largo plazo centrado en el desarrollo de nuevos biomateriales a partir de ELRs que van desde hidrogeles bioactivos a nanopartículas demostrando su versatilidad en diversas aplicaciones biomédicas. Se estudia un amplio conjunto de interesantes propiedades: biocompatibilidad, propiedades mecánicas, comportamiento termosensible y la capacidad de autoensamblaje. Se han obtenido hidrogeles químicamente entrecruzados (microestructurados o porosos) como soportes celulares para ingeniería de tejidos, diferentes nano-objetos autoensamblados en medio acuoso y finalmente mediante autoensamblado en condiciones fisiológicas se obtendrán geles inyectables, los cuales son muy útiles en terapias quirúrgicas no invasivas y medicina regenerativaDepartamento de Física de la Materia Condensada, Cristalografía y Mineralogí

Recent Contributions of Elastin-Like Recombinamers to Biomedicine and Nanotechnology

Abstract: The emergence of the new scientific field known as nanomedicine is being catalyzed by multiple improvements in nanoscience techniques and significant progress in materials science, especially as regards the testing of novel and sophisticated biomaterials. This conjuncture has furthered the development of promising instruments in terms of detection, bioanalysis, therapy, diagnostics and imaging. Some of the most innovative new biomaterials are protein-inspired biomimetic materials in which modern biotechnology and genetic-engineering techniques complement the huge amount of information afforded by natural protein evolution to create advanced and tailor-made multifunctional molecules. Amongst these protein-based biomaterials, Elastin-like Recombinamers (ELRs) have demonstrated their enormous potential in the fields of biomedicine and nanoscience in the last few years. This broad applicability derives from their unmatched properties, particularly their recombinant and tailor-made nature, the intrinsic characteristics derived from their elastin-based origin (mainly their mechanical properties and ability to self-assemble as a result of their stimuli-responsive behavior), their proven biocompatibility and biodegradability, as well as their versatility as regards incorporating advanced chemical or recombinant modifications into the original structure that open up an almost unlimited number of multifunctional possibilities in this developing field. This article provides an updated review of the recent challenges overcome by using these recombinant biomaterials in the fields of nano- and biomedicine, ranging from nanoscale applications in surface modifications and self-assembled nanostructures to drug delivery and regenerative medicine.Este trabajo forma parte de Proyectos de Investigación financiados por la Comisión Europea a través del Fondo Europeo de Desarrollo Regional (ERDF), por el del MINECO (MAT2010-15982, MAT2010-15310, PRI-PIBAR-2011-1403 and MAT2012-38043), la Junta de Castilla y León (VA049A11, VA152A12 y VA155A12) y el Instituto de Salud Carlos III bajo el Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León

Hydrogel-based logic circuits for planar microfluidics and lab-on-a-chip automation

The transport of vital nutrient supply in fluids as well as the exchange of specific chemical signals from cell to cell has been optimized over billion years of natural evolution. This model from nature is a driving factor in the field of microfluidics, which investigates the manipulation of the smallest amounts of fluid with the aim of applying these effects in fluidic microsystems for technical solutions. Currently, microfluidic systems are receiving attention, especially in diagnostics, \textit{e.g.} as SARS-CoV-2 antigen tests, or in the field of high-throughput analysis, \textit{e.g.} for cancer research. Either simple-to-use or large-scale integrated microfluidic systems that perform biological and chemical laboratory investigations on a so called Lab-on-a-Chip (LoC) provide fast analysis, high functionality, outstanding reproducibility at low cost per sample, and small demand of reagents due to system miniaturization. Despite the great progress of different LoC technology platforms in the last 30 years, there is still a lack of standardized microfluidic components, as well as a high-performance, fully integrated on-chip automation. Quite promising for the microfluidic system design is the similarity of the Kirchhoff's laws from electronics to predict pressure and flow rate in microchannel structures. One specific LoC platform technology approach controls fluids by active polymers which respond to specific physical and chemical signals in the fluid. Analogue to (micro-)electronics, these active polymer materials can be realized by various photolithographic and micro patterning methods to generate functional elements at high scalability. The so called chemofluidic circuits have a high-functional potential and provide “real” on-chip automation, but are complex in system design. In this work, an advanced circuit concept for the planar microfluidic chip architecture, originating from the early era of the semiconductor-based resistor-transistor-logic (RTL) will be presented. Beginning with the state of the art of microfluidic technologies, materials, and methods of this work will be further described. Then the preferred fabrication technology is evaluated and various microfluidic components are discussed in function and design. The most important component to be characterized is the hydrogel-based chemical volume phase transition transistor (CVPT) which is the key to approach microfluidic logic gate operations. This circuit concept (CVPT-RTL) is robust and simple in design, feasible with common materials and manufacturing techniques. Finally, application scenarios for the CVPT-RTL concept are presented and further development recommendations are proposed.:1 The transistor: invention of the 20th century 2 Introduction to fluidic microsystems and the theoretical basics 2.1 Fluidic systems at the microscale 2.2 Overview of microfluidic chip fabrication 2.2.1 Common substrate materials for fluidic microsystems 2.2.2 Structuring polymer substrates for microfluidics 2.2.3 Polymer chip bonding technologies 2.3 Fundamentals and microfluidic transport processes 2.3.1 Fluid dynamics in miniaturized systems 2.3.2 Hagen-Poiseuille law: the fluidic resistance 2.3.3 Electronic and microfluidic circuit model analogy 2.3.4 Limits of the electro-fluidic analogy 2.4 Active components for microfluidic control 2.4.1 Fluid transport by integrated micropumps 2.4.2 Controlling fluids by on-chip microvalves 2.4.3 Hydrogel-based microvalve archetypes 2.5 LoC technologies: lost in translation? 2.6 Microfluidic platforms providing logic operations 2.6.1 Hybrids: MEMS-based logic concepts 2.6.2 Intrinsic logic operators for microfluidic circuits 2.7 Research objective: microfluidic hydrogel-based logic circuits 3 Stimuli-responsive polymers for microfluidics 3.1 Introduction to hydrogels 3.1.1 Application variety of hydrogels 3.1.2 Hydrogel microstructuring methods 3.2 Theory: stimuli-responsive hydrogels 3.3 PNIPAAm: a multi-responsive hydrogel 4 Design, production and characterization methods of hydrogel-based microfluidic systems 4.1 The semi-automated computer aided design approach for microfluidic systems 4.2 The applied design process 4.3 Fabrication of microfluidic chips 4.3.1 Photoresist master fabrication 4.3.2 Soft lithography for PDMS chip production 4.3.3 Assembling PDMS chips by plasma bonding 4.4 Integration of functional hydrogels in microfluidic chips 4.4.1 Preparation of a monomer solution for hydrogel synthesis 4.4.2 Integration methods 4.5 Effects on hydrogel photopolymerization and the role of integration method 4.5.1 Photopolymerization from monomer solutions: managing the diffusion of free radicals 4.5.2 Hydrogel adhesion and UV light intensity distribution in the polymerization chamber 4.5.3 Hydrogel shrinkage behavior of different adhesion types 4.6 Comparison of the integration methods 4.7 Characterization setups for hydrogel actuators and microfluidic measurements . 71 4.7.1 Optical characterization method to describe swelling behavior 4.7.2 Setup of a microfluidic test stand 4.8 Conclusion: design, production and characterization methods 5 VLSI technology for hydrogel-based microfluidics 5.1 Overview of photolithography methods 5.2 Standard UV photolithography system for microfluidic structures 5.3 Self-made UV lithography system suitable for the mVLSI 5.3.1 Lithography setup for the DFR and SU-8 master exposure 5.3.2 Comparison of mask-based UV induced crosslinking for DFR and SU-8 5.4 Mask-based UV photopolymerization for mVLSI hydrogel patterning 5.4.1 Lithography setup for the photopolymerization of hydrogels 5.4.2 Hydrogel photopolymerization: experiments and results 5.4.3 Troubleshooting: photopolymerization of hydrogels 5.5 Conclusion: mVLSI technologies for hydrogel-based LoCs 6 Components for chemofluidic circuit design 6.1 Passive components in microfluidics 6.1.1 Microfluidic resistor 6.1.2 Planar-passive microfluidic signal mixer 6.1.3 Phase separation: laminar flow signal splitter 6.1.4 Hydrogel-based microfluidic one-directional valves 6.2 Hydrogel-based active components 6.2.1 Reversible hydrogel-based valves 6.2.2 Hydrogel-based variable resistors 6.2.3 CVPT: the microfluidic transistor 6.3 Conclusion: components for chemofluidic circuits 7 Hydrogel-based logic circuits in planar microfluidics 7.1 Development of a planar CVPT logic concept 7.1.1 Challenges of planar microfluidics 7.1.2 Preparatory work and conceptional basis 7.2 The microfluidic CVPT-RTL concept 7.3 The CVPT-RTL NAND gate 7.3.1 Circuit optimization stabilizing the NAND operating mode 7.3.2 Role of laminar flow for the CVPT-RTL concept 7.3.3 Hydrogel-based components for improved switching reliability 7.4 One design fits all: the NOR, AND and OR gate 7.5 Control measures for cascaded systems 7.6 Application scenarios for the CVPT-RTL concept 7.6.1 Use case: automated cell growth system 7.6.2 Use case: chemofluidic converter 7.7 Conclusion: Hydrogel-based logic circuits 8 Summary and outlook 8.1 Scientific achievements 8.2 Summarized recommendations from this work Supplementary information SI.1 Swelling degree of BIS-pNIPAAm gels SI.2 Simulated ray tracing of UV lithography setup by WinLens® SI.3 Determination of the resolution using the intercept theorem SI.4 Microfluidic master mold test structures SI.4.1 Polymer and glass mask comparison SI.4.2 Resolution Siemens star in DFR SI.4.3 Resolution Siemens star in SU-8 SI.4.4 Integration test array 300 μm for DFR and SU-8 SI.4.5 Integration test array 100 μm for SU-8 SI.4.6 Microfluidic structure for different technology parameters SI.5 Microfluidic test setups SI.6 Supplementary information: microfluidic components SI.6.1 Compensation methods for flow stabilization in microfluidic chips SI.6.2 Planar-passive microfluidic signal mixer SI.6.3 Laminar flow signal splitter SI.6.4 Variable fluidic resistors: flow rate characteristics SI.6.5 CVPT flow rate characteristics for high Rout Standard operation proceduresDer Transport von lebenswichtigen Nährstoffen in Flüssigkeiten sowie der Austausch spezifischer chemischer Signale von Zelle zu Zelle wurde in Milliarden Jahren natürlicher Evolution optimiert. Dieses Vorbild aus der Natur ist ein treibender Faktor im Fachgebiet der Mikrofluidik, welches die Manipulation kleinster Flüssigkeitsmengen erforscht um diese Effekte in fluidischen Mikrosystemen für technische Lösungen zu nutzen. Derzeit finden mikrofluidische Systeme vor allem in der Diagnostik, z.B. wie SARS-CoV-2-Antigentests, oder im Bereich der Hochdurchsatzanalyse, z.B. in der Krebsforschung, besondere Beachtung. Entweder einfach zu bedienende oder hochintegrierte mikrofluidische Systeme, die biologische und chemische Laboruntersuchungen auf einem sogenannten Lab-on-a-Chip (LoC) durchführen, bieten schnelle Analysen, hohe Funktionalität, hervorragende Reproduzierbarkeit bei niedrigen Kosten pro Probe und einen geringen Bedarf an Reagenzien durch die Miniaturisierung des Systems. Trotz des großen Fortschritts verschiedener LoC-Technologieplattformen in den letzten 30 Jahren mangelt es noch an standardisierten mikrofluidischen Komponenten sowie an einer leistungsstarken, vollintegrierten On-Chip-Automatisierung. Vielversprechend für das Design mikrofluidischer Systeme ist die Ähnlichkeit der Kirchhoff'schen Gesetze aus der Elektronik zur Vorhersage von Druck und Flussrate in Mikrokanalstrukturen. Ein spezifischer Ansatz der LoC-Plattformtechnologie steuert Flüssigkeiten durch aktive Polymere, die auf spezifische physikalische und chemische Signale in der Flüssigkeit reagieren. Analog zur (Mikro-)Elektronik können diese aktiven Polymermaterialien durch verschiedene fotolithografische und mikrostrukturelle Methoden realisiert werden, um funktionelle Elemente mit hoher Skalierbarkeit zu erzeugen.\\ Die sogenannten chemofluidischen Schaltungen haben ein hohes funktionales Potenzial und ermöglichen eine 'wirkliche' on-chip Automatisierung, sind jedoch komplex im Systemdesign. In dieser Arbeit wird ein fortgeschrittenes Schaltungskonzept für eine planare mikrofluidische Chiparchitektur vorgestellt, das aus der frühen Ära der halbleiterbasierten Resistor-Transistor-Logik (RTL) hervorgeht. Beginnend mit dem Stand der Technik der mikrofluidischen Technologien, werden Materialien und Methoden dieser Arbeit näher beschrieben. Daraufhin wird die bevorzugte Herstellungstechnologie bewertet und verschiedene mikrofluidische Komponenten werden in Funktion und Design diskutiert. Die wichtigste Komponente, die es zu charakterisieren gilt, ist der auf Hydrogel basierende chemische Volumen-Phasenübergangstransistor (CVPT), der den Schlüssel zur Realisierung mikrofluidische Logikgatteroperationen darstellt. Dieses Schaltungskonzept (CVPT-RTL) ist robust und einfach im Design und kann mit gängigen Materialien und Fertigungstechniken realisiert werden. Zuletzt werden Anwendungsszenarien für das CVPT-RTL-Konzept vorgestellt und Empfehlungen für die fortlaufende Entwicklung angestellt.:1 The transistor: invention of the 20th century 2 Introduction to fluidic microsystems and the theoretical basics 2.1 Fluidic systems at the microscale 2.2 Overview of microfluidic chip fabrication 2.2.1 Common substrate materials for fluidic microsystems 2.2.2 Structuring polymer substrates for microfluidics 2.2.3 Polymer chip bonding technologies 2.3 Fundamentals and microfluidic transport processes 2.3.1 Fluid dynamics in miniaturized systems 2.3.2 Hagen-Poiseuille law: the fluidic resistance 2.3.3 Electronic and microfluidic circuit model analogy 2.3.4 Limits of the electro-fluidic analogy 2.4 Active components for microfluidic control 2.4.1 Fluid transport by integrated micropumps 2.4.2 Controlling fluids by on-chip microvalves 2.4.3 Hydrogel-based microvalve archetypes 2.5 LoC technologies: lost in translation? 2.6 Microfluidic platforms providing logic operations 2.6.1 Hybrids: MEMS-based logic concepts 2.6.2 Intrinsic logic operators for microfluidic circuits 2.7 Research objective: microfluidic hydrogel-based logic circuits 3 Stimuli-responsive polymers for microfluidics 3.1 Introduction to hydrogels 3.1.1 Application variety of hydrogels 3.1.2 Hydrogel microstructuring methods 3.2 Theory: stimuli-responsive hydrogels 3.3 PNIPAAm: a multi-responsive hydrogel 4 Design, production and characterization methods of hydrogel-based microfluidic systems 4.1 The semi-automated computer aided design approach for microfluidic systems 4.2 The applied design process 4.3 Fabrication of microfluidic chips 4.3.1 Photoresist master fabrication 4.3.2 Soft lithography for PDMS chip production 4.3.3 Assembling PDMS chips by plasma bonding 4.4 Integration of functional hydrogels in microfluidic chips 4.4.1 Preparation of a monomer solution for hydrogel synthesis 4.4.2 Integration methods 4.5 Effects on hydrogel photopolymerization and the role of integration method 4.5.1 Photopolymerization from monomer solutions: managing the diffusion of free radicals 4.5.2 Hydrogel adhesion and UV light intensity distribution in the polymerization chamber 4.5.3 Hydrogel shrinkage behavior of different adhesion types 4.6 Comparison of the integration methods 4.7 Characterization setups for hydrogel actuators and microfluidic measurements . 71 4.7.1 Optical characterization method to describe swelling behavior 4.7.2 Setup of a microfluidic test stand 4.8 Conclusion: design, production and characterization methods 5 VLSI technology for hydrogel-based microfluidics 5.1 Overview of photolithography methods 5.2 Standard UV photolithography system for microfluidic structures 5.3 Self-made UV lithography system suitable for the mVLSI 5.3.1 Lithography setup for the DFR and SU-8 master exposure 5.3.2 Comparison of mask-based UV induced crosslinking for DFR and SU-8 5.4 Mask-based UV photopolymerization for mVLSI hydrogel patterning 5.4.1 Lithography setup for the photopolymerization of hydrogels 5.4.2 Hydrogel photopolymerization: experiments and results 5.4.3 Troubleshooting: photopolymerization of hydrogels 5.5 Conclusion: mVLSI technologies for hydrogel-based LoCs 6 Components for chemofluidic circuit design 6.1 Passive components in microfluidics 6.1.1 Microfluidic resistor 6.1.2 Planar-passive microfluidic signal mixer 6.1.3 Phase separation: laminar flow signal splitter 6.1.4 Hydrogel-based microfluidic one-directional valves 6.2 Hydrogel-based active components 6.2.1 Reversible hydrogel-based valves 6.2.2 Hydrogel-based variable resistors 6.2.3 CVPT: the microfluidic transistor 6.3 Conclusion: components for chemofluidic circuits 7 Hydrogel-based logic circuits in planar microfluidics 7.1 Development of a planar CVPT logic concept 7.1.1 Challenges of planar microfluidics 7.1.2 Preparatory work and conceptional basis 7.2 The microfluidic CVPT-RTL concept 7.3 The CVPT-RTL NAND gate 7.3.1 Circuit optimization stabilizing the NAND operating mode 7.3.2 Role of laminar flow for the CVPT-RTL concept 7.3.3 Hydrogel-based components for improved switching reliability 7.4 One design fits all: the NOR, AND and OR gate 7.5 Control measures for cascaded systems 7.6 Application scenarios for the CVPT-RTL concept 7.6.1 Use case: automated cell growth system 7.6.2 Use case: chemofluidic converter 7.7 Conclusion: Hydrogel-based logic circuits 8 Summary and outlook 8.1 Scientific achievements 8.2 Summarized recommendations from this work Supplementary information SI.1 Swelling degree of BIS-pNIPAAm gels SI.2 Simulated ray tracing of UV lithography setup by WinLens® SI.3 Determination of the resolution using the intercept theorem SI.4 Microfluidic master mold test structures SI.4.1 Polymer and glass mask comparison SI.4.2 Resolution Siemens star in DFR SI.4.3 Resolution Siemens star in SU-8 SI.4.4 Integration test array 300 μm for DFR and SU-8 SI.4.5 Integration test array 100 μm for SU-8 SI.4.6 Microfluidic structure for different technology parameters SI.5 Microfluidic test setups SI.6 Supplementary information: microfluidic components SI.6.1 Compensation methods for flow stabilization in microfluidic chips SI.6.2 Planar-passive microfluidic signal mixer SI.6.3 Laminar flow signal splitter SI.6.4 Variable fluidic resistors: flow rate characteristics SI.6.5 CVPT flow rate characteristics for high Rout Standard operation procedure

Modification of Behavior of Elastin-like Polypeptides by Changing Molecular Architecture

Elastin-like polypeptides (ELP) are environmentally responsive polymers that exhibit phase separation in response to external stimuli such as temperature, pH, light, and ionic strength. It has been shown that the sequence of the pentapeptide, its length, and the solution concentration are very important in the transition of the molecules from soluble to insoluble, but there has not been any detailed study of the effect of molecular architecture on the behavior of ELPs.In this study we designed, synthesized and characterized ELPs with different architectures and chemical identities to probe the effect of molecular design on the microscopic and macroscopic behavior of ELP molecules and to compare them to the linear ELP molecules. These new architectures also helped us better understand the theory of folding and aggregation of ELPs. The design was based on constructing three-armed star molecules by tagging a trimer forming oligomerization domain to the ELP chains. ELPs were chosen to have different chemical identities by changing the pentapetide sequence. The molecules were synthesized by molecular biology techniques and characterized by different methods.Our results show that capping the three ELP chains forces the chains to fold into more extended rod-like constructs prior to aggregation. A mathematical model was developed to predict the behavior of ELP chains at the transition temperature and it was shown that there is a difference between N- and C- terminal capping ELPs seem to fold at lower temperatures when their N-termini are held together. It was also shown that the constructs with both their ends capped can be designed such that they fold into a stable unit at much lower temperatures than the linear constructs without necessarily aggregation at higher temperatures. The trimer constructs were also used to make micellar aggregates that were characterized by dynamic and static light scattering. It was shown that the size of the micelles can be controlled by adjusting salt concentration or by making mixtures

Functional surface microstructures inspired by nature – From adhesion and wetting principles to sustainable new devices

In the course of evolution nature has arrived at startling materials solutions to ensure survival. Investigations into biological surfaces, ranging from plants, insects and geckos to aquatic animals, have inspired the design of intricate surface patterns to create useful functionalities. This paper reviews the fundamental interaction mechanisms of such micropatterns with liquids, solids, and soft matter such as skin for control of wetting, self-cleaning, anti-fouling, adhesion, skin adherence, and sensing. Compared to conventional chemical strategies, the paradigm of micropatterning enables solutions with superior resource efficiency and sustainability. Associated applications range from water management and robotics to future health monitoring devices. We finally provide an overview of the relevant patterning methods as an appendix

Functional surface microstructures inspired by nature : From adhesion and wetting principles to sustainable new devices

In the course of evolution nature has arrived at startling materials solutions to ensure survival. Investigations into biological surfaces, ranging from plants, insects and geckos to aquatic animals, have inspired the design of intricate surface patterns to create useful functionalities. This paper reviews the fundamental interaction mechanisms of such micropatterns with liquids, solids, and soft matter such as skin for control of wetting, self-cleaning, anti-fouling, adhesion, skin adherence, and sensing. Compared to conventional chemical strategies, the paradigm of micropatterning enables solutions with superior resource efficiency and sustainability. Associated applications range from water management and robotics to future health monitoring devices. We finally provide an overview of the relevant patterning methods as an appendix

Nanostructured functional multilayer coatings incorporating biomimetic macromolecules for biomedical applications

Tese de doutoramento do Programa Doutoral em Engenharia BiomédicaThe 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.A modificação de superfícies tem sido um aspeto fundamental em biologia e biotecnologia, em aplicações como a expansão de células, desenvolvimento de biomateriais e preparação de substratos para medicina regenerativa. Neste trabalho, a técnica de camada-a-camada foi utilizada na modificação de superfícies para vários fins, como filmes com adesividade e adesão celular melhoradas, cápsulas para administração de drogas, e o posicionamento espacial magnético. A hipótese foi a de que esta técnica poderia ser utilizada na modificação de superfícies planares ou tridimensionais, usando exclusivamente polímeros obtidos de várias origens naturais ou a partir de vias recombinantes. O quitosano (CHI), um reconhecido policatião com origens marinhas, foi recorrentemente utilizado como ingrediente em várias formulações de multicamadas, impulsionado por forças intermoleculares, tais como interações eletrostáticas e hidrofóbicas. Primeiro, revestimentos de CHI e um exopolissacarídeo bacteriano adesivo, levano, foram construídos. Como controlo, foram comparados com filmes de CHI e alginato – polissacarídeos marinhos considerados bons adesivos naturais. A força adesiva de 50 bicamadas de CHI/levano foi determinada como sendo cerca de 3 vezes mais elevada do que o controlo. A adesão de células L929 foi também maior em filmes contendo levano. Em seguida, CHI foi utilizado juntamente com recombinâmeros tipo-elastina (ELRs), uma classe de polímeros tipo-elastina, exibindo a sequência de adesão celular “arginina-glicina-ácido aspártico” (RGD). Embora interações eletrostáticas tenham sido invocadas para a construção de multicamadas auto-organizadas, estas não foram o único mecanismo de interação entre CHI e ELRs. A construção de filmes de CHI e um de nove ELRs diferentes em conteúdo aminoacídico, tamanho e biofuncionalidade foi monitorizado in situ a pH 4.0 e 5.5 utilizando uma microbalança de cristais de quartzo. As suas espessuras foram estimadas usando o modelo de Voigt para filmes viscoelásticos, revelando que os filmes mais espessos foram obtidos na presença de interações hidrofóbicas entre ELRs e CHI parcialmente neutralizado, isto é, quando o pH foi ajustado para 5.5, próximo do seu pKa. Filmes de ELR/alginato também foram monitorados, completando um total de 36 combinações estudadas. Os resultados obtidos estiveram em concordância com os dados dos filmes baseados em CHI: filmes mais espessos foram obtidos para alginato parcialmente neutralizado, a um valor de pH de 4.0. Através de medições de ângulos de contato, demonstrou-se que os revestimentos bidimensionais de CHI e ELR-RGD eram responsivos a estímulos: exibiram uma superfície moderadamente hidrofóbica mas converteram-se em extremamente hidrofílicas quando se aumentou a temperatura, o pH ou força iónica acima de 50ºC, 11, ou 1,25 M, respetivamente. A presença de RGD melhorou a adesão de células SaOs-2, em comparação com filmes terminados ou em CHI ou num análogo não bioativo, RDG. Os revestimentos de CHI/ELR-RGD foram extrapolados para a terceira dimensão sob a forma de microcápsulas esféricas construídas em torno de partículas de carbonato de cálcio. Com a finalidade de administração de drogas, foram estudados dois mecanismos: (i) a difusão simples para o exterior das microcápsulas e (ii) a incorporação celular. No primeiro caso, estudos de libertação a 25 e 37°C com microcápsulas contendo albumina do soro bovino demonstraram uma libertação sustentada ao longo de 14 dias, mas sendo as cápsulas menos permeáveis a uma temperatura fisiológica, devido à transição molecular dos ELRs induzida pela temperatura. Além disso, um número mais elevado de camadas proporcionou uma barreira adicional à difusão da proteína encapsulada. As microcápsulas foram também não citotóxicas para células L929. No segundo caso, a eficácia de internalização e tráfego intracelular de microcápsulas funcionalizadas com RGD ou a sequência não bioativa RDG por células estaminais mesenquimais humanas (hMSCs) foi avaliada. Os dados sugerem que as microcápsulas foram internalizadas principalmente através de macropinocitose, e que a funcionalização da superfície não desempenhou um papel significativo neste fenómeno, embora o processamento intracelular tenha sido mais rápido para microcápsulas funcionalizadas com RGD. Ambos os tipos de microcápsulas foram não citotóxicas para hMSCs para rácios de microcápsula/célula entre 5:1 e 100:1. Finalmente, com inspiração na hierarquia complexa e estrutura compartimentada das células, esferas macroscópicas de alginato liquefeito revestidas com camadas de CHI/alginato foram concebidas. Dentro deste ambiente liquefeito, rodamina e microcápsulas foram confinadas, com estas últimas podendo conter ou mais rodamina ou nanopartículas magnéticas (MNPs). A 25 e 37ºC, rodamina quando encapsulada no interior de microcápsulas mostrou um perfil de libertação sustentada, sendo a cinética de difusão ainda mais reduzida a 25°C. A libertação de rodamina encapsulada no compartimento externo de alginato não exibiu diferenças significativas em função da temperatura. MNPs encapsuladas dentro das microcápsulas providenciaram resposta magnética a todo o dispositivo compartimentado e capacidade de mobilidade dirigida. O trabalho aqui desenvolvido mostra que a técnica de modificação camada-a-camada é uma técnica versátil capaz de fornecer meios para desenvolver uma ampla gama de sistemas úteis em aplicações biomédicas, desde suportes planos adesivos e para adesão celular até estruturas tridimensionais para a administração “inteligente” de drogas, terapias guiadas e microreatores personalizados para o desenvolvimento de modelos de doenças e produção de microtecidos em laboratório