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

New applications of dynamic combinatorial chemistry to medicinal chemistry

Die Verwendung dynamisch kombinatorischer Chemie (DCC) in medizinisch-chemischen Projekten kann eine sehr hilfreiche Strategie sein, um Anknüpfungspunkte für die Wirkstoffentdeckung zu finden. 14-3-3 Proteine spielen eine Rolle in verschiedenen Krankheiten und vielen biologischen Prozessen. Proteine dieser Familie beteiligen sich an Protein-Protein-Interaktionen (PPIs) und können die Aktivität der Bindungspartner sowohl hoch- als auch herabregulieren. Eine andere Familie relevanter Targets sind die Glukansucrasen, welche wichtige Enzyme in der Initiierung und Entwicklung von kariogenen dentalen Biofilmen, allgemein bekannt als Plaque, sind. In den letzten beiden Kapiteln wurde Endothiapepsin für Protein-vermittelte DCC (ptDCC) verwendet. Endothiapepsin gehört zur Familie der Aspartylproteasen, welche zum Beispiel an der Reifung des HIV Viruspartikels beteiligt sind. Im Verlauf dieser Arbeit fokussieren wir uns auf die Anwendung von DCC in verschiedenen Projekten. Die Hauptleistungen sind: 1) die Beschreibung des hausinternen DCC-Protokolls, in welchem Aspekte wie Löslichkeit von Bausteinen und Produkten, Proteinstabilität und weiteres wichtige zu beachten sind, 2) die Anwendung von Acylhydrazon-basierter DCC auf zwei Targets, eine Glukansucrase und ein PPI-Target, 3) die Identifikation kleiner Moleküle, die PPIs von 14-3-3/ Synaptopodin stabilisieren, 4) die Erweiterung des Reaktionsspielraums der ptDCC durch zwei zusätzliche Reaktionen: Nitron- und Thiazolidinbildung.Applying dynamic combinatorial chemistry (DCC) to medicinal chemistry projects can be a helpful strategy for finding starting points in the drug-discovery process. As relevant drug target, 14-3-3 proteins play a role in several diseases and many biological processes. Proteins of this family engage in protein-protein interactions (PPIs), and can up-or down-regulate their binding partner’s activity. Another family of relevant targets are glucansucrases, which are important enzymes in the initiation and development of cariogenic dental biofilms, commonly known as dental plaque. In the last two chapters, endothiapepsin was used for protein-templated DCC (ptDCC). Endothiapepsin belongs to the family of the aspartic proteases, which are involved in for example the maturation of the HIV virus particle. Throughout this thesis, we focus on applying DCC to various projects. The main achievements are: 1) the description of the in-house protocol of DCC, in which aspects like solubility of building blocks and products, protein stability and more need to be taken in to account, 2) the application of acylhydrazone-based DCC to two targets, a (PPI)-target and a glucansucrase, 3) the identification of small-molecules, which stabilise PPIs of 14-3-3/ synaptopodin, 4) expanding the reaction toolbox of ptDCC by two additional reactions: nitrone and thiazolidine formation

Nanobiotechnologie: Werkzeuge für die Proteomik : molekulare Organisation und Manipulation von Proteinen und Proteinkomplexen in Nanodimensionen

First milestone of this Ph.D. thesis was the successful extension of conventional NTA/His-tag technique to self-assembling, multivalent chelator thiols for high-affinity recognition as well as stable and uniform immobilization of His-tagged proteins on chip surfaces. Bis-NTA was linked via an oligoethylene glycol to alkyl thiols by an efficient modular synthesis strategy yielding a novel, multivalent compound for formation of mixed SAMs with anti-adsorptive matrix thiols on gold. Multivalent chelator chips allow a specific, high-affinity, reversible, long-term immobilization of His-tagged proteins. In AFM studies reversibility of the specific protein immobilization process was visualized at single molecule level. The entire control over the orientation of the immobilized protein promotes this chip surface to an optimal platform for studies focusing on research targets at single molecule level and nanobiotechnology. Based on the constructed protein chip platform above and a novel AFM mode (contact oscillation mode, COM) – developed during the current Ph.D. work – protein nanolithography under physiological conditions enabling fabrication of active biomolecular patterns in countless variety has been established. Reversible COM-mediated nanostructuring is exceptionally suitable for multiplexed patterning of protein assemblies in situ. The first selfassembled protein layer acts as a biocompatible and ductile patterning material. Immobilized proteins can be replaced by the AFM tip applying COM, and the generated structures can be erased and refilled with different proteins, which are immobilized in a uniform and functional manner. Multi-protein arrays can be systematically fabricated by iterative erase-and-write processes, and employed for protein-protein interaction analysis. Fabrication of two-dimensionally arranged nanocatalytic centres with biological activity will establish a versatile tool for nanobiotechnology. As an alternative chip fabrication approach, the combined application of methodologies from surface chemistry, semiconductor technology, and chemical biology demonstrated successfully how pre-patterned templates for micro- and nanoarrays for protein chips are fabricated. The surface physical, as well the biophysical experiments, proved the functionality of this technology. The promises of such process technology are fast and economic fabrication of ready-to-use nanostructured biochips at industrial scale. Membrane proteins are complicated in handling and hence require sophisticated solutions for chip technological application. A silicon-on-insulator (SOI) chip substrate with microcavities and nanopores was employed for first technological investigation to construct a protein chip suitable for membrane proteins. The formation of an artificial lipid bilayer using vesicle fusion on oxidized SOI cavity substrates was verified by CLSM. Future AFM experiments will give further insights into the chip architecture and topography. This will provide last evidence of the sealing of the cavity by the lipid bilayer. Transmembrane proteins will be employed for reconstitution experiments on this membrane protein chip platform. Highly integrated microdevices will find application in basic biomedical and pharmaceutical research, whereas robust and portable point-of-care devices will be used in clinical settings.Erster Meilenstein der vorliegenden Arbeit war die erfolgreiche Erweiterung des konventionellen NTA/His-tag-Konzepts auf selbst-assemblierende, multivalente Chelatorthiole für die hochaffine Erkennung und stabile, einheitliche Immobilisierung His-getaggter Proteine auf Chipoberflächen. Mittels einer effizienten, modularen Synthesestrategie wurden Bis-NTA-Module über Oligoethylenglykoleinheiten an Alkylthiole angebunden. Diese Chelatorthiole wurden zusammen mit antiadsorptiven Matrixthiolen zur Ausbildung gemischter selbst-assemblierender Monolagen (SAMs) auf Goldoberflächen eingesetzt. Die multivalenten Chelatorchips erlauben eine spezifische, hochaffine, umkehrbare und langfristige Immobilisierung His-getaggter Proteine. Die Umkehrbarkeit der spezifischen Proteinimmobilisierung wurde in rasterkraftmikroskopischen (AFM) Studien bis zur Einzel-Molekül-Ebene visualisiert. Die vollständige Kontrolle über die Orientierung immobilisierter Proteine qualifiziert diese entwickelte Chipoberfläche zu einer optimalen Plattform für Anwendungsbereiche der Einzelmolekülbiochemie und Nanobiotechnologie. Basierend auf dieser Plattform für Proteinchips und einem – im Rahmen dieser Arbeit – neuentwickelten AFM-Modus (Kontaktoszillationsmodus, COM) wurde die „Protein-Nanolithographie“ etabliert, welche die Fabrikation von aktiven, biomolekularen Strukturen in unzähliger Vielfalt ermöglicht. Die umkehrbare COM-vermittelte Nanolithographie ist insbesondere für die multiplexe Anordnung von Proteinverbänden in situ geeignet. Die erste Schicht immobilisierter Proteine fungiert als ein biokompatibles und verformbares Strukturierungsmaterial. Diese immobilisierten Proteine können nun im Kontaktoszillationsmodus mit der AFM-Spitze lokal entfernt („Löschen“) und gegen andere Proteine – die an die freigelegte Chipoberfläche ebenfalls spezifisch und funktional immobilisieren – ausgetauscht werden („Schreiben“). Arrays, bestehend aus mehreren unterschiedlichen Proteinen können nun systematisch in iterativen Lösch-und-Schreib-Vorgängen fabriziert und für Proteininteraktionsanalysen eingesetzt werden. Die Fabrikation von zwei-dimensional arrangierten nanokatalytischen Zentren mit biologischer Aktivität wird von großem Nutzen für die Nanobiotechnologie sein. Eine alternative Herstellungsmethode aus einer Kombination von Oberflächenchemie, Halbleitertechnologie und chemischer Biologie wurde für die Fabrikation von vorstrukturierten Templaten für Mikro- und Nanoarrays entwickelt. Die Funktionalität dieser Chipplattform wurde anhand oberflächen- und biophysikalischer Experimente erfolgreich gezeigt. Zukünftiges Ziel ist die Anfertigung vorstrukturierter Template in der Dimension weniger Nanometer zur Ausbildung von Bio-Arrays mit einzelnen Molekülen. Ein weiteres Ziel besteht in der kompletten Verlagerung des Herstellungsprozesses in die Gasphase. Eine Produktion in der Gasphase verspricht eine schnelle und wirtschaftliche Erzeugung sofort einsatzbereiter nanostrukturierter Biochips im industriellen Maßstab. Der Umgang mit Membranproteinen verlangt besondere Vorkehrungen im experimentellen Milieu, ebenso speziell sind die Bedürfnisse in den entsprechenden Chip-Anwendungen. Ein Chip mit Mikrokavitäten und Nanoporen, basierend auf der „Silicon-on-Insulator“ (SOI)-Technologie, wurde für erste technologische Studien zum Entwurf eines Proteinchips für Membranproteine eingesetzt. Künstliche Lipidmembranen wurden auf der SOI-Oberfläche mittels Vesikelfusion ausgebildet und mit konfokaler Laser-Scanning-Mikroskopie gezeigt. Zukünftige AFM-Experimente werden weitere Einsichten in die Chiparchitektur und Topographie ermöglichen. Transmembranproteine werden in Rekonstitutionsexperimenten für funktionale Studien der Membranproteinchips eingesetzt. Anwendungsbereiche solcher hochintegrierten Mikrosysteme sind sowohl in der biologischen Grundlagenforschung als auch in mobilen Diagnostikgeräten im klinischen Einsatz zu finden

Multifunctional Polymer Materials: From Synthesis to Disinfection

Polymer materials have wide applications in many industries, such as the food, pharmacy, construction, textile, and cosmetics industries. For the past few years, polymer materials have drawn the attention of scientists and engineers as a good disinfectant due to their advanced manufacturing methods, large surface areas, good stability, and lowcost. More importantly, polymer materials can be functionalized with various chemical groups to increase their affinity towards microorganisms and broaden their applications. In this thesis, four types of multifunctional polymer materials were synthesized to investigate their disinfection ability on bacterial cells.By using molecular imprinting technology, a small molecule-chloramphenicol-imprinted polymer material of nanometer size was prepared via precipitation polymerization, and large bacteria-imprinted polymer materials of micrometer size were synthesized via surface imprinting-Pickering emulsion polymerization. Both materials had highly specific binding to the targeted template and could be used as adsorbents. In precipitation polymerization, 3-(acrylamido)phenylboronic acid was added to introduce boronic acid on the material surface. In neutral and basic aqueous solutions, boronic acid groups formed reversible boronate ester bonds with the cis-diol groups of the polysaccharides on bacterial surfaces. The release of chloramphenicol led to a high antibiotic concentration around the bacterial cells, which killed the cells. In Pickering emulsion polymerization, positively charged vinyl-functionalized polyethylenimine self-assembled with negatively charged bacterial cells and acted as a stabilizer for the emulsion. Therefore, bacteria-recognition sites based on the bacteria’s physical property formed on the surface of polymer beads after crosslinking polymerization. Ag+ was released from the preloaded hydrophobic Ag nanoparticles in the polymer beads to deactivate the bound bacterial cells.To realize multifunctional materials for antibacterial applications, nanometer sized polymer materials were prepared with glycidyl methacrylate by precipitation polymerization and microemulsion polymerization. The epoxide groups were opened by polyethylenimine, which was further used to stabilize Ag nanoparticles. The final material selfassembled with bacterial cells via electrostatic interactions. The amino groups and Ag nanoparticles endowed the composite material with disinfection ability. The molecular spectra of bacteria could also be acquired via surfaceenhanced Raman scattering from the surface Ag nanoparticles.In addition to spherical polymer materials, temperature tunable deactivation polymers were also synthesized with (methacryloyloxy)ethyl]trimethylammonium by atom transfer radical polymerization, which was initiated by an initiator containing a boronic acid group. By further modification of the terminal alkyl bromide, a fluorescent molecule,fluorescein 5(6)-isothiocyanate, was added to the polymer chain. The obtained polymers self-assembled with bacterial cells via reversible boronate ester bonds and electrostatic interactions. At 40 ℃, the polymers showed effective deactivation of bacterial cells via a synergistic effect. At 20 ℃, the polymers displayed lower or no toxicityto bacterial cells and could be used to label bacterial cells in flow cytometry and fluorescence imaging