Dissertations / Theses on the topic 'DENDRIMERIC PEPTIDES'

Photodynamic therapy (PDT) is an emerging therapy for the treatment of cancer and various other human disorders. 5-Aminolaevulinic acid (ALA) is a simple natural product that is of great interest for PDT because it can be converted within cells via the haem biosynthetic pathway to the photosensitiser, protoporphyrin IX (PpIX). ALA-PDT has become a first line clinical approach for the treatment of cancerous and precancerous skin lesions (e.g Bowen’s disease, basal skin carcinomas, and actinic keratosis) that would otherwise require significant conventional surgery. However, ALA being a zwitterion suffers from poor lipid solubility and at the same time has stability issues at physiological or alkaline pH. The work herein describes some novel strategies to enhance the delivery of ALA to specific cell types using targeted ALA dendrimeric prodrugs. Specifically, it describes the synthesis of molecules consisting of branched units with 3 or more copies of ALA attached to a central core structure (e.g. gallic acid) using copper-catalysed azide-alkyne click chemistry (CuAAC). Selective delivery of the dendrimeric ALA cargo was achieved by attachment of a homing peptide to an independently addressable functional group on the prodrug core. As proof of concept of this approach, systems were prepared containing a peptide that allows selective targeting of the epidermal growth factor receptor (EGFR) which is overexpressed in a variety of tumours. Targeted ALA delivery and PpIX production was studied with these prodrugs in EGFR-expressing breast adenocarcinoma cells (MDA-MB-231 cells) and a peptide-targeted derivative with 9 ALA units was found to have enhanced PDT efficacy compared to an equimolar dose of ALA. Other targeting units that have been attached to these dendrimeric ALA prodrugs include biomolecules such as vitamin E, thymidine (a nucleoside) and a glucose derivative. Additionally, strain-promoted azide-alkyne cycloadditions (SPAAC) of the same EGFR-targeting peptide with some classical photosensitisers were also investigated and biological studies in EGFR-overexpressing cell lines were carried out. Lastly, a group of cell penetrating peptide-ALA conjugates have been synthesised via CuAAC as a novel approach for targeted ALA delivery.

Elastin like peptides (ELPs)—derived from the protein elastin—are widely used as thermoresponsive components in biomaterials due to their LCST (lower critical solution temperature) behavior at a characteristic transition temperature (Tt). While linear ELPs have been well investigated, few reports focused on branched ELPs. Using lysine (Lys, with an additional side-chain amine) as branching units, ELP dendrimers were synthesized by solid-phase peptide synthesis (SPPS) with up to 155 amino acid residues. A secondary structure change with decreasing ratio of random coil and increasing ratio of β-turn upon heating, which is typical of linear ELPs, was confirmed by circular dichroism spectroscopy for all peptides. Conformational change did not show evident dependence on topology, while a higher Tt was observed for dendritic peptides than for their linear control peptides with the same number of GLPGL repeats. Variable-temperature small-angle X-ray scattering (SAXS) measurements showed a size increase and fractal dimension upon heating, even below the Tt. These results were further confirmed by cryogenic transmission electron microscopy (cryo-TEM), and micro differential scanning calorimetry (micro-DSC), revealing the presence of aggregates below the Tt. These results indicated the presence of a pre-coacervation step in the LCST phase transition of the ELP dendrimers. We further prepared hydrogels by crosslinking hyaluronic acid (HA) with ELP dendrimers. We invesigated their physical properties with scanning electron microscopy (SEM), swelling tests, SAXS, and model drug loading/release experiments. Most of the HA_denELP hydrogels retained transparent upon gelation, but after lyophilization and reswelling remained opaque for days. This reswelling process was carefully investigated with time-course SAXS studies, and was attributed to forming pre-coacervates in the gelation step, which slowly reswelled during rehydration. We then prepared hydrogels with H2S-releasing aroylthiooxime (SATO) groups and showed human neutrophil elastase-responsive H2S-releasing properties with potential applications in treating chronic diseases with recurring inflammation. Furthermore, we prepared a series of wedge-shaped triblock polyethylene glycol (PEG)-ELP dendrimer-C16 (palmitic acid) conjugate amphiphiles with adjustable Tts. Various techniques were used to investigate their hierarchical structures. The triblock PEG-peptide-C16 conjugate amphiphiles were thermoresponsive and showed a morphology change from small micelles to large aggregates. However, the hydrophilic shell and strong tendency for micelle formation limited the thermoresponsive assembly, leading to slow turbidity change in the LCST transition. The secondary structure was twisted from conventional β-sheet, and the thermoresponsive trend observed in typical ELP systems was not observed, either. Variable temperature NMR showed evidence for coherent dehydration of the PEG and ELP segments, probably due to the relatively short blocks. Utilizing the micelles with hydrophobic cavity, we were able to encapsulate hydrophobic drugs, with promising applications for localized drug release in hyperthermia.
Doctor of Philosophy

WHO has reported that antibiotic resistance is the third major cause of human death all over the globe. Recent study, has focused on the development of new antibacterial resistance drugs. Herein, we tried to synthesis a series of polymers that can mimic the HDPs. HDPs can target the bacterial cell membrane and they have less chances to develop bacterial resistance. We synthesized the amphiphilic polycarbonates that are highly selective to Gram-positive bacteria, including multidrug resistant pathogens. The membrane disruption activity of these polymers was proved by fluorescence and TEM studies and the drug resistance study showed that the polymers don’t develop bacterial resistance. In order to further design the molecules that can target a broad spectrum of bacteria, we have designed a series of lipidated dendrimers that can target the Gram-positive and Gram-negative bacteria. These dendrimers mimic the HDPs and target the bacterial cell membrane. Dendrimers are reported to inhibit the formation of bacterial biofilm which makes them promising for their future development of antibiotic agents. Apart from the synthesis of polymers and dendrimers as antibacterial agents, we have designed a series of small molecular antibacterial agents that are based on the acylated reduced amide scaffold and small dimeric cyclic guanidine derivatives. These molecules display good potency against a panel of multidrug-resistant Gram-positive and Gram-negative bacterial strains. Meanwhile, they also effectively inhibit the biofilm formation. Mechanistic studies suggest that these compounds kill bacteria by compromising bacterial membranes, a mechanism analogous to that of host-defense peptides (HDPs). Lastly, we also demonstrate that these molecules have excellent in vivo activity against MRSA in a rat model. This class of compounds could lead to an appealing class of antibiotic agents combating drug-resistant bacterial strains.

La malaltia d’Alzheimer es caracteritza per l’acumulació de plaques extracel.lulars amiloides, formades pels anomenats pèptids amiloides (Aβ40). L’homeòstasi del coure (Cu) es veu afectada en l’etiologia de la malaltia d’Alzheimer, encara que el seu rol és un fet controvertit. Per a l’estudi de la influència del Cu(II) en l’agregació del pèptid amiloide, la morfologia i l’estructura secundària dels agregats amiloides formats en presència de Cu(II) s’han utilitzat AFM, TEM, SEM, SAXS, FTIR i espectrometria de fluorescència. A més, els efectes tòxics d’aquests agregats s’han estudiat en cèl.lules neuronals. Els resultats obtinguts mostren que aquests agregats són amorfos i presenten una toxicitat més alta que les fibres. Per a l’estudi dels dendrímers de maltosa com a possibles moduladors de l’agregació i de la toxicitat del pèptide amiloide. S’ha confirmat que aquests dendrimers no són tòxics en cèl.lules neuronals i que són capaços de modular l’agregació i toxicitat del pèptid amiloide. Aquests resultats permeten considerar als dendrimers de maltosa com a possibles eines per a reduir la toxicitat dels pèptids amilodies.
Senile plaques of Alzheimer’s disease patients are composed primarily of the amyloid-β-peptide (Aβ). Recent studies implicate Cu(II) in the aetiology of AD. The role of Cu(II) in ADis currently highly disputed. Influence of Cu(II) on Aβ aggregation and amyloidogenic properties of glycodendrimers were investigated in this thesis. AFM, TEM, SEM, SAXS, FTIR and fluorescence spectroscopy were used to study a morphology and a secondary structure of Aβ-Cu(II) aggregates. The toxic effects of Aβ40-Cu(II) amorphous aggregates was confirmed for neuronal cell lines. It was shown that maltose glycodendrimers can be efficiently used to modulate Alzheimer’s amyloid peptide aggregation and inhibit cell toxicity by facilitating the clustering of amyloid fibrils. These results show that glycodendrimers are promising non-toxic agents in the search for anti-amyloidogenic compounds. It was also suggested that fibril clumping may be anti-amyloid toxicity strategy.

Inspiriert von den natürlich vorkommenden Antibiotika der Gramicidin Familie und ihrer d-(alt)-l Aminosäuresequenz, die es diesen Oligopeptiden ermöglicht, eine beta–helikale Sekundärstruktur einzunehmen, war das Hauptziel dieser Arbeit die Synthese und Charakterisierung von Peptiden und diversen Pseudopeptiden mit regulärer all-l und d-(alt)-l Sequenz und die Untersuchung des Einflusses dieser stereochemischen Variation auf die Strukturen und Eigenschaften dieser Verbindungen. Zusätzlich ergab der Austausch von Amid-Bindungen im Peptid-Rückgrat durch verschiedene Isostere diverse, teils einzigartige Pseudopeptid-Strukturen, wohingegen Verzweigung des linearen Peptid-Rückgrates zu sphärischen Molekülen führte. Alle Projekte zielten auf die Entwicklung und Synthese diskreter Oligomere für Strukturuntersuchungen, sowie auf die Einbindung der jeweiligen Strukturelemente in Polymere. Die Polymerization geeigneter Monomere zu Polymeren soll zu makro- und supramolekularen Nano-Objekten führen. Die divergent/konvergente Synthese einer Serie von Oligo-d-(alt)-l-lysinen zielte auf die Generierung hydrophiler, pH-sensitiver nanotubularer Strukturen. Schrittweiser Austausch von Amid-Bindungen des Peptid-Rückgrates durch Ester-(alt)-Urea-Einheiten führte zu all-l und d-(alt)-l Oligopseudoleucinen mit 50% und 0% Amid-Bindungs-Anteil. Design, Synthese und Polymerisation von AB-“Click”-Monomeren, basierend auf all-l and l-(alt)-d lysin Dipeptiden, ergaben hochmolekulare, Triazol-enthaltende Polypseudopeptide, deren Seitenketten mit Pyrenbuttersäure quantitativ postfunktionalisiert werden konnten. Die Einführung von Verzweigung in Glutamat-Peptide ergab chirale Dendrimere mit adressierbaren fokalen und periphären Funktionalitäten, sowie variabler Ladungsdichte. Design, Synthese und Polymerisation eines Glutamat basierenden AB2-“Click”-Monomers lieferte verwandte chirale hyperverzweigte Polypseudopeptide.
Inspired by the naturally occurring antibiotics of the Gramicidin family and their d-(alt)-l amino acid sequence, enabling these oligopeptides to adopt a beta–helical secondary structure, the work presented in this thesis targeted the syn-thesis and characterization of peptides and diverse pseudopeptides with regular all-l and d-(alt)-l sequences and the influence of this stereochemical variation on the compounds’ structures and properties. Further diversification of the struc-tures as obtained by replacing amide bonds in the peptide backbone with differ-ent isosteres, affording unique pseudopeptide structures. In addition spherical molecules were generated by introducing branching into the linear peptide scaf-fold. Throughout all projects, the aim was the design and synthesis of discrete oligomers for structural investigations and the incorporation of the respective structural elements into polymers via the polymerization of suitable monomers, in order to generate nanoscale macromolecular and supramolecular objects. The divergent/convergent synthesis of a series of oligo-d-(alt)-l-lysines targeted the generation of hydrophilic, pH-sensitive nanotubular structures. The stepwise replacement of peptide backbone amide bonds with ester-(alt)-urea moieties afforded all-l and d-(alt)-l oligopseudoleucines with 50% and 0% amide content. The design, synthesis, and polymerization of an AB-“Click”-monomer, based on all-l and l-(alt)-d lysine dipeptides afforded high molecular weight, triazole con-taining polypseudopeptides. Quantitative coupling to pyrene butyric acid afforded the respective side chain labeled polymers. The introduction of branching into glutamate peptides afforded fully chiral den-drimers with addressable focal and peripheral functionalities and variable charge density. The design, synthesis, and polymerization of a glutamate based AB2-“Click”-monomer led to related chiral hyperbranched polypseudopeptides.

RNA interference (RNAi) is a conservative post-transcriptional gene silencing mechanism that can be mediated by small interfering RNAs (siRNAs). Given the effectiveness and specificity of RNAi, the administration of siRNA molecules is a promising approach to cure diseases caused by abnormal gene expression. However, as siRNA is susceptible to degradation by nucleases and it can hardly penetrate cell membranes due to its polyanionic nature, a successful translation of the RNAi mechanism for therapeutic purposes is contingent on the development of safe and efficient delivery systems. This dissertation described the development of novel siRNA delivery systems on the basis of polymeric and dendrimeric materials and also demonstrated the application of one optimized delivery system to deliver therapeutic siRNAs in a cardiovascular disease model in vivo. We studied a linear peptide polymer made from cell penetrating peptide monomers and investigated the contribution of the polymeric structure, degradability, and ligand conjugation to the siRNA loading capacity, biocompatibility, and transfection efficiency of polymeric materials. With the obtained knowledge and experience, we invented a neutral crosslinked delivery system aiming to solve the inherent drawbacks of traditional cationic delivery systems that are based on electrostatic interactions. The new concept utilized buffering amines to temporarily bind siRNA and a crosslinking reaction to immobilize the formed particles, and targeting ligands modified on the neutral dendrimer surface further enhanced the interactions between the delivery vehicles and target cells. The obtained delivery system allowed stability, safety, controllability, and targeting ability for siRNA delivery, and the method developed here could be transformed to other polymeric or dendrimeric cationic materials to make them safer and more efficient. To exploit the therapeutic potential of siRNA delivery, we developed a tadpole-shaped dendrimeric material to deliver siRNA against an Angiotensin II receptor in a rat ischemia-reperfusion model. Our results showed that the nonaarginine-conjugated tadpole dendrimer was capable of delivering siRNA effectively to cardiac cells both in vitro and in vivo, and the successful down-regulation of the Angiotensin II receptor preserved the cardiac functions and reduced the infarct size post-myocardial infarction. This dissertation paves a way for transforming multifunctional non-viral siRNA delivery systems into potent therapeutic strategies for the management of cardiovascular diseases.

Cancer is lacking early detection methods and treatment specificity. In order to increase the sensitivity and specificity towards cancer cells, we propose the use of multivalent interactions targeting specific receptor combinations at the cancer cell surface. In this thesis, we explored the design of multimers, which could provide such interactions. The design was investigated and revisited based on specific parameters, essential for the creation of multivalent interactions such as thermodynamics. The synthesis was designed so that libraries of homo- and hetero-multimers of different valencies can be obtained efficiently with good yields. The established synthetic scheme is empowered by its modularity, necessary to investigate different essential factors. Trimers composed of micromolar affinity MSH(4) targeting the MC1-R, overexpressed in melanoma, were investigated on a model cell line and resulted in the creation of nanomolar affinity constructs with up to 350 fold increase in affinity. Different multimers such as hexavalent and nonavalent dendrimers were synthesized and studied for their properties. All constructs had nanomolar affinity and showed to be non-toxic up to micromolar concentrations and imaging studies also confirmed their internalization, which overall demonstrate the potential for these compounds to be used as markers for cancer cells and as delivery agents. Trimers targeting the CCK2-R were similarly investigated for their potential as pancreatic cancer markers. However, those constructs did not seem to result in the expected enhancements in affinity, but the affinity of the initial monovalent agonist was in the 10-50 nanomolar range. As we were unable to design micromolar affinity agonist we investigated the use of antagonists. This study, revealed the importance of thermodynamics in the creation of multivalent interaction. Heterotrivalent ligands (CCK and MSH) were investigated for their potential in cross-linking different receptors and the study demonstrated the subtility to detect cross-linking. Finally, the different attempts toward the efficient synthesis of a tetra-orthogonal scaffold, a key feature needed to generate multimers that could target up to 3 different receptors was investigated and showed promising results. It is our hypothesis that such an approach will ultimately lead to specific markers of tumor cells, which could be used as diagnosis agents when modified with an imaging moiety and as a therapeutic agent when modified with a drug.

Les dendrimères sont très prometteurs du fait de leur structure unique et de leur multivalence. Cependant, leur synthèse souffre de problèmes de défauts de structure et de présence de produits secondaires très similaires. Des approches synthétiques alternatives sont donc fortement désirées. L'objectif de ma thèse consiste à explorer la synthèse sur support solide et l’approche d'autoassemblage pour la préparation de dendrimères.La première partie de ma thèse se concentre sur la synthèse de dendrimères en phase solide. Nous avons tout d'abord développé une méthode de synthèse pour les dendrimères poly(amidoamines) basée sur la chimie des peptides. Nous avons ensuite construit une petite bibliothèque de dendrimères de type triazine en faisant varier la taille et la terminaison des dendrimères pour créer une variété de dendrimères. Nous avons aussi tenté de synthétiser des dendrimères poly(aminoesters) bien que nous n'ayons pu les obtenir du fait du caractère labile de ces dendrimères.La deuxième partie de ma thèse vise à appliquer l’approche d'autoassemblage pour la construction de dendrimères supramoléculaires comme théranostiques combinant l'imagerie et la thérapie. Nous avons synthétisé un petit dendrimère amphiphile portant DOTA pour chélater le Gd (III). Ce dendrimère est capable de s'autoassembler en supramolécule et d’encapsuler l’agent anticancéreux doxorubicine, pour construire des agents théranostiques à base de dendrimères multivalents.L’ensemble de ma thèse se consacre au développement de stratégies en phase solide et de l'autoassemblage pour construire des dendrimères pour les applications dans les domaines biomédicaux et des matériaux
Dendrimers hold great promise for wide applications thanks to their unique structural architecture and multivalent cooperativity. However, dendrimer synthesis often suffers from structural defects caused by incomplete reactions and difficulties associated with purification. Consequently, alternative synthetic approaches to overcome the limitations of current dendrimer synthesis are in high demand.My first PhD project mainly focuses on establishing novel strategies and methodologies for solid-phase dendrimer synthesis with advantages of convenient complete synthesis and easy purification procedures. We first developed a new and concise solid-phase synthesis of PAMAM dendrimers based on the adoption of peptide synthesis chemistry. We then constructed a small library of triazine dendrimers varying in generations and surface groups with a view to rapidly synthesizing dendrimers with structural diversity. We also strived to synthesize poly(aminoester) dendrimers although we had difficult to get it thorough.My second PhD program aims to apply the self-assembly approach for constructing supramolecular dendrimer theranostics. A small DOTA-conjugated amphiphilic dendrimer with Gd(III)-chelation was synthesized and self-assembled into supramolecular nanomicelles to encapsulate the anticancer drug doxorubicin. The obtained system constitutes a multivalent nanotheranostic to combine imaging purpose with therapeutic utility.In summary, my PhD program mainly contributes to elaborating strategies for dendrimer synthesis using both solid-phase method and self-assembly approach in the view to realizing and broadening their applications in the arenas of biomedical and material sciences

Bacterial infections have posed a serious threat to the public health due to the significant rise of the infections caused by antibiotic-resistant bacteria. There has been considerable interest in the development of antimicrobial agents which mimic the natural HDPs, and among them biodegradable polymers are newly discovered drug candidates with ease of synthesis and low manufacture cost compared to synthetic host defense peptides. Herein, we present the synthesis of biocompatible and biodegradable polymers including polycarbonate polymers, unimolecular micelle hyperbranched polymers and dendrimers that mimic the antibacterial mechanism of HDPs by compromising bacterial cell membranes. The developed amphiphilic polycarbonates are highly selective to Gram-positive bacteria, including multidrug-resistant pathogens and the unimolecular micelle hyperbranched polymers showed promising broad-spectrum activity. However, lipidated amphiphilic dendrimers with low molecular weight display potent and selective antimicrobial activity against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. In addition to antibacterial activity against planktonic bacteria, these dendrimers were also shown to inhibit bacterial biofilms effectively. These class of polymers may lead to a useful generation of antibiotic agents with practical applications.

The objectives of this research were to identify the individual ionization constants (pKa values) of lipopeptide (daptomycin), evaluate the factors of pH, concentration, temperature, and calcium ions on daptomycin aggregation in aqueous solutions, and elucidate the effects of conformation and aggregation on ionization and the interaction mechanism between polyamidoamine (PAMAM) dendrimers and daptomycin. Daptomycin is a cyclic anionic lipopeptide antibiotic. It is composed of 13 amino acids with six ionizable groups, four side-chain carboxylic acids and two side-chain amine residues. The pKa values for individual daptomycin residues have not been elucidated. The sequence-specific pKa values for the four acidic residues and one aromatic amine (Kyn-13) in daptomycin were determined in the monomeric state by TOCSY 2D 1H NMR. From the NMR pH titration, the estimated pKa values for Asp-3, Asp-9, and mGlu-12 were determined to be 4.15, 3.85, and 4.55 in the absence of salt, and 4.07, 3.83, and 4.39 in the presence of 150 mM NaCl, respectively. The pKa value for Asp-7 is estimated to be ~1.01 in the absence of salt and 1.31 in the presence of salt. The estimated Hill coefficients for Asp-7 were 0.72 and 1.31 in the absence and presence of salt, respectively. The increase in Hill coefficients from 0.72 to 1.31 with increasing salt concentration is consistent with the estimated lower pKa in the absence of salt and suggests that a salt bridge is formed in solution possibly between Asp-7 acidic group and the neighboring Orn-6 basic group. The pKa value of the aromatic amine (Kyn-13) was confirmed using UV and fluorescence spectroscopic titrations. Aggregation behavior and critical aggregation concentration (CAC) values of daptomycin were evaluated in the different pH aqueous solutions by using the complementary analytical techniques, fluorescence, dynamic and static light scattering, and NMR spectroscopy. Based on fluorescence resonance energy transfer (FRET) from donor Trp-1 to acceptor Kyn-13, the CAC values were determined by an upward inflection of the intrinsic fluorescence emission from Kyn-13 at 460 nm as a function of increasing daptomycin concentration. The pH-dependent CAC values were determined to be 0.14 mM at pH 3.0, 0.12 mM 4.0, and 0.20 mM at pH 2.5 and 5.0. The CAC values obtained by fluorescence spectroscopy were confirmed by dynamic light scattering and NMR spectroscopy. The effects of temperature and calcium ion on daptomycin aggregation were also discussed. The interaction mechanism between daptomycin and PAMAM dendrimers generation 5 and 6 was studied using fluorescence spectroscopy. The shapes of binding isotherms daptomycin were quantitatively described by one- and two-site binding models to estimate binding capacity and dissociation constants. Both solvent pH values and PAMAM generation size were shown to affect the binding model and parameters. The interaction between daptomycin and PAMAM dendrimer was proposed wherein the ionized Asp-3 and Asp-9 residues of daptomycin interact with PAMAM cationic surface amine.

Thesis (Ph. D.)--Ohio State University, 2005.
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Tese de mestrado, Bioquímica, Universidade de Lisboa, Faculdade de Ciências, 2009
Dendrimers are a family of branched compounds that share a common layout where wedges emerge radially from a core by means of a regular branching pattern. Peptide dendrimers are a specific kind of dendrimers formed by alternating functional amino acids with branching diamino acids. There has been increasing interest in the synthesis of peptide-based dendritic architectures modelling specific aspects of biological function. Some results are already available, demonstrating these molecules ability to act as enzyme models and to mimic natural ligands. Unfortunately, most studies concerning peptide dendrimers lack structural information at the molecular level. The theoretical study published so far, reported peptide dendrimers presenting shapes close to spheres, though experimental studies on the same systems suggest the existence of more disordered states. Herein, we characterize five third-generation peptide dendrimers (B1, B1H, B1HH, B1HHH and C1) through multiple long molecular dynamics simulations (MD), and analyse their conformational details and folding preferences in solution. Special emphasis is placed on the analysis of conformational trends representative of the examined models. The conformational sampling results, obtained through MM/MD simulations, were scrutinized using several approaches. Namely, histogram analysis, phi-psi dihedral distributions, inter-residue distance matrices, shape analysis and principal coordinate analysis. The adequacy of each approach to discern the conformation space of peptide dendrimers is discussed. Using these analysis procedures we were able to observe two distinct types of behaviour (sphere-like and bowl-like structures), both asserting the enormous structural flexibility characterizing these molecules; and the myriad of conformational states available to them. Our conclusions can be interpreted together with the available experimental results, contributing to a synergistic understanding of the structure-function relation in peptide dendrimers, and casting the bases for novel knowledge-based applications.
Os dendrímeros são uma família de compostos ramificados que partilham uma arquitectura comum, onde diferentes cadeias emergem radialmente de um mesmo núcleo (ou centro) através de um padrão de ramificação regular. Os dendrímeros peptídicos, são uma classe particular de dendrímeros, constituída por estruturas que incorporam de forma alternada e iterativa resíduos de amino-ácidos funcionais (resíduos de espaçamento) com resíduos de diamino-ácidos ramificados (resíduos de ramificação). Os resíduos de diamino-ácidos ramificados promovem a bifurcação das cadeias peptídicas e a aquisição da estrutura dendrítica. A possibilidade de sintetizar dendrímeros com composições que mimetizem as funções de moléculas biológicas, constitui o aliciante para a investigação neste campo cientifico. Em particular, a síntese planeada e controlada de estruturas dendríticas baseadas nos componentes apresentados pelas moléculas biológicas, como peptidos ou glícidos, constitui um desafio atractivo pelas potenciais aplicações que dai podem emergir. De facto, já foram reportados dendrímeros peptídicos que modelam aspectos específicos de funções biológicas, tais como: modelos enzimáticos para catálise dirigida (“enzimas artificiais”); mimetização de co-factores naturais (de que e exemplo a vitamina B12); transportadores de fármacos, pois quando acopladas aos ligandos adequados estas moléculas tem a capacidade de aderir à membrana celular. É também importante referir que várias destas moléculas têm sido estudadas enquanto modelos de folding das proteínas naturais, pois investigações experimentais indiciam que alguns dendrímeros peptídicos podem apresentar, em solução, uma estrutura compacta semelhante à das proteínas globulares. Contudo, a maioria dos estudos experimentais realizados até à data são omissos no que concerne a informação estrutural, e carecem do enquadramento adequado a nível molecular e atómico. O único estudo teórico publicado sobre dendrímeros peptídicos parece confirmar a ideia de que, em solução estas moléculas apresentam, de facto, formas semelhantes a esferas, isto apesar de existirem evidências experimentais que sugerem a existência de estados conformacionais mais desordenados, nesses mesmos sistemas. Considerando o grande interesse que estas moléculas tem vindo a despertar, a verdade é que pouco se sabe sobre o seu arranjo estrutural tridimensional, e sobre os processos que a ele conduzem (folding). Nesta tese tentamos preencher algumas destas lacunas. Para tal, procedemos à caracterização de cinco dendrímeros peptídicos de terceira geração (que designamos por B1, B1H, B1HH, B1HHH e C1) com diferentes constituintes peptídicos. Os sistemas que escolhemos como objecto de estudo, estão directamente relacionados com a coordenação da aquocobalamina (análogo da vitamina B12) a dendrímeros peptídicos, ainda que apenas três deles tenham sido sintetizados e caracterizados experimentalmente (B1, B1H e C1). Deste modo, pretendemos não só investigar as suas preferências conformacionais, mas também inferir possíveis relações entre a sua estrutura e a capacidade para desempenhar uma função análoga à das moléculas biológicas (transcobalamina). É importante salientar que de entre os dendrímeros que foram sintetizados experimentalmente, e que são também aqui estudados, os que apresentam maior capacidade de coordenação com a aquacobalamina, são os que possuem um menor número de resíduos com potencial de coordenação. Este aparente paradoxo é por si só interessante e pode estar interligada com aspectos mais estruturais. Como temos por objectivo compreender as alterações e a variabilidade subjacentes às estruturas tridimensionais dos diferentes dendrímeros, empregamos metodologias adequadas ao detalhe da escala que pretendemos investigar. Nomeadamente, métodos computacionais de simulação molecular (MM/MD). Optámos portanto por simular cada um destes cinco sistemas através de múltiplas e longas simulações de dinâmica molecular, utilizando a água enquanto solvente explícito. Com efeito, no trabalho que conduziu a esta tese, realizamos simulações que contabilizam aproximadamente 1 μs-1 para cada um dos dendrímeros em estudo. No que respeita a estes sistemas, isto é muito superior ao tempo simulado em estudos anteriores. Nas últimas décadas a investigacao científica tem beneficiado imenso do avanço das técnicas de simulação computacional, que providenciam resultados e formas de escrutinar sistemas, que são de outra forma normalmente inacessíveis. A dinâmica molecular, especificamente, permite “seguir” a evolução temporal dos átomos que constituem um sistema, através da integração das equações de Newton para o movimento de corpos. É inclusive um dos métodos computacionais de eleição para estudar fenómenos biomoleculares. Os resultados obtidos com esta técnica de amostragem conformacional permitiram-nos analisar e identificar de forma adequada, os detalhes estruturais de cada um dos dendrímeros peptídicos. Colocamos especial ênfase nos arranjos estruturais mais estáveis. As conformações tridimensionais obtidas a partir das trajectórias resultantes das simulações, foram agrupadas de forma a obtermos os ensembles conformacionais característicos de cada dendrímero. Sobre estes conjuntos de conformações realizamos várias análises. Começamos por investigar algumas das propriedades que caracterizam estes sistemas, como o raio de giração, o número total de ligações de hidrogénio, a distância máxima entre os dois átomos mais afastados de cada estrutura, a superfície acessível ao solvente, entre outros. O raio de giração revelou ser a propriedade que individualmente, melhor espelha as variações intrínsecas a estes sistemas. Adicionalmente, procedemos também a caracterização da distribuição dos valores de phi-psi característicos dos diedros de cada um dos dendrímeros. Complementamos esta análise com o estudo das matrizes que reflectem as distãncias mínimas entre os resíduos de todas as conformações. Posteriormente aplicamos metodologias de análise conformacional que envolvem a determinação da energia livre, associada a diferentes coordenadas reaccionais (ou de folding) para cada estrutura nos diferentes ensembles, obtendo assim as correspondentes superfícies energéticas (folding landscapes). Utilizamos esta abordagem por forma a obter folding landscapes bi- e tridimensionais. Em especifíco, utilizamos como coordenadas de folding os valores do raio de giração, do root mean square deviation (RMSD), dos componentes principais do tensor do raio de giração diagonalizado, e os valores para a posição relativa das diferentes conformações, num espaço concordante com a matriz de RMSD, utilizando para tal o método de análise das coordenadas principais (PCoorA). Utilizando o tensor do raio de giração, foi possível investigar a forma dos arranjos estruturais de cada dendrímero peptídico, tendo inclusive sido definido um espaço tridimensional baseado nos componentes principais do tensor diagonalizado (espaço de giração). A capacidade de cada uma destas abordagens para discriminar de forma adequada o espaço das conformações dos dendrímeros peptídicos é discutida ao longo da tese. Dos diversos procedimentos de análise conformacional empregues, resulta uma clara indicação de que, em solução, os dendrímeros peptídicos podem apresentar dois comportamentos preferenciais distintos: estruturas compactas que privilegiam as interacções entre os diferentes resíduos, semelhantes a esferas (sphere-like); e estruturas “abertas” com as diferentes ramificações espaçadas, em que as interacções entre resíduos não adjacentes são minimizadas, semelhantes a taças (bowl-like). Ambas estas configurações atómicas consubstanciam a enorme flexibilidade estrutural que parece caracterizar estas moléculas, dando provas da miríade de estados conformacionais que lhes estão acessíveis. Foi ainda possível verificar a existência de evidências que suportam a ideia de que estas moléculas possuem uma grande robustez estrutural. Isto é, pequenas alterações na composição dos resíduos de amino-ácidos que as constituem não parecem desencadear alterações conformacionais significativas nos arranjos estruturais preferenciais. Através da comparação entre o coeficiente de difusao experimental disponível para um dos dendrímeros, e o coeficiente de difusão calculado com base nas trajectórias obtidas por simulação, foi possível verificar que os modelos utilizados, reflectem de forma adequada os sistemas experimentais. Concluiu-se também que o campo de força (force field) GROMOS 53A6 possui a capacidade de transferabilidade apropriada para lidar com estas moléculas. Os pontos fortes e fracos dos nossos modelos são discutidos ao longo da tese. Durante este trabalho foi ainda desenvolvida e implementada uma metodologia que permite o cálculo eficiente do RMSD entre estruturas dendríticas. As conclusões apresentadas nesta tese podem ser interpretadas juntamente com os resultados experimentais disponíveis, de forma a contribuir para uma compreensão sinérgica da relação entre a estrutura e a função dos dendrímeros peptídicos, lançando as fundações para aplicações inovadoras.

Dendrimers are a family of highly branched synthetic compounds that share a common layout where wedges emerge radially from a core by means of a regular branching pattern. Topologically, dendrimers are characterized by three distinct regions: core, branches, and periphery, with the latter being typically composed of functionalized end-groups.

碩士
高雄醫學大學
醫藥暨應用化學研究所
96
This project focus on the design, preparation and characterization of carrier macromolecular which are able to carry pyridoxal-5-phosphate (PLP). We had synthesized several modified PAMAM dendrimers. With synthetic short peptide containing lysine, we introduce those peptides on the surface of G3 and G4 PAMAM dendrimer. As far, compounds 14, 39 and 41 were successfully prepared under linear assembly approach. Compounds 14 and 39 are derivatives of G3 and G4 PAMAM dendrimer, respectively. To learn the binding affinity of synthetic dendrimer, the Scatchard-Klotz analysis was applied. The result shows the binding constant of our synthetic molecle up to 0.35 mM-1 which is similar to the known natural PLP associate enzymes. Meanwhile, the compound 41, tyrosine was used instead of phenylalanine, to reveal the possible capality of tyrosine. The results suggest our hypothesis should be reasonable and feasible.

碩士
高雄醫學大學
醫藥暨應用化學研究所
98
Pyridoxal 5′-Phosphate (PLP) is the active member of of vitamin B6. PLP are known to perform numbers of reactivities in a variety of enzymes in which the lysine is a conserved residue for harboring PLP via Schiff base moiety. This is also known as external aldimine. During the course of reaction, the inbound substrate will form new Schiff base with PLP, and known as external aldimine. The exchange between external and internal aldimine is important for the demonstration of reactions. Base on the previous experimental results, we design a tripeptide involving lysine to modify the surface of PAMAM dendrimer for binding the Pyridoxal 5′-Phosphate. The designed peptides are Phe-Lys-X. The aromatic ring of phenylamine enhances the binding through PLP by?n???{???ninteraction. By the same reason, histidine, tryptophan, or tyrosine are chosen to be the third residue. During the synthesis of peptide, we found the protecting group is crucial to the solubility of those tripeptides. Those with Fmoc protecting group exhibit poor solubility. (G; 4, 5, 7)-dendri-PAMAM-(APO-Phe-Lys)n was selected for the investigation of rasemization. Under basic condition, the racemization was monitered by HPLC analysis. This result proves the ability of those synthetic dendrimers as catalyst of racemization.

碩士
高雄醫學大學
醫藥暨應用化學研究所
97
There are many surface groups on the dendrimers surfaces. Histidine accesses the good binding affinity of metal ions, such as copper and nickel ions. In this thesis, we modified the surface groups of PAMAM dendrimer with different number of histidine peptide. The designed peptides were prepared by solid phase peptide synthesis (SPPS). Meanwhile, we modified the PAMAM dendrimer with histidine residue successfully, and the ability to bind copper was confirmed through the electrochemistry. In the future, these compounds may be a good metal binder. Meanwhile, prostate cancer is one of the top ten cancer incidence. The most important way to improve the drugs solubility and diminish side effect is to modify the drugs with specific peptides. For the prostate cancer, we choose the Bombesin (BBN) as our target peptides, the sequences are QWAVGHLM. At first, we synthesized the final compounds on solid phase, but the drug was decomposed in TFA which is necessary to collect peptide from resin. Thereafter, we have to synthesize and purify the peptide first then to modify the drug with the peptide. With this, we compared the solubility before and after the peptide modification by UV detection.

Novel manganese(I) tricarbonyl complexes based on the tridentate bis(pyrazolyl)ethylamine (bpea) ligand with pendant functionalized phenyl groups were synthesized and conjugated to biological carrier systems like peptides and dendrimers. Their dark stability establishes them as CORM prodrugs. The monomers show a faster CO-release compared to the peptide and dendrimer conjugates. However, both monomers and peptide conjugates release two equivalents of CO upon photoactivation at 365 nm. The dendrimer conjugates can deliver up to seven equivalents of CO due to the higher number of Mn(CO)3 moieties per molecular unit. In the future, the biological activity of the conjugates needs to be further explored to establish the targeted delivery of CO to cells and tissues
Das Ziel dieser Doktorarbeit war die Synthese von Mangan(I)-Tricarbonylkomplexen als neuartige photoaktivierbare CO-releasing molecules (PhotoCORMs) und ihrer Peptid- und Dendrimer-Konjugate als Trägersysteme für das targeted delivery von Kohlenstoffmonoxid in biologischen Systemen. Dafür wurde eine Serie von sechs Mn(I)-Tricarbonylkomplexen basierend auf dem Bis(pyrazolyl)ethylamin-Liganden (bpea) hergestellt welche einen para-substituierten Phenylring mit einer peripheren Iod- oder Alkin-Gruppe enthalten. Diese sollten eine Konjugation an Biomoleküle mittels Sonogashira- oder CuAAC-"click"-Reaktion ermöglichen. Alle Verbindungen wurden in guter Ausbeute mit hoher Reinheit erhalten. Die Einkristall-Röntgenstrukturen der Verbindungen mit Iod- und Alkin- Substituenten belegen die faciale Koordination des tridentaten bpea-Liganden an die Mn(CO)3-Gruppe. Alle Komplexe zeigen bei Lichtausschluß eine sehr gute Stabilität in Dimethylsulfoxid-Lösung über bis zu 14 h, die Photoaktivierung bei 365 nm führt dagegen zur Freisetzung von zwei Äquivalenten Kohlenstoffmonoxid pro Mol Komplex. Obwohl die Verbindungen MLCT-Banden um 350 nm ausweisen, können sie auch noch bei 410 nm stimuliert werden, was für biologische Anwendungen vorteilhaft ist. Die photoinduzierte CO-Freisetzung wurde auch mittels IR- Spektroskopie in Lösung verfolgt. Die Banden neu auftretender Intermediate konnten dabei mit Hilfe von DFT-Rechnungen zugeordnet werden und belegen die Bildung von cis-Mn(CO)2-Spezies nach der Freisetzung eines ersten Äquivalents Kohlenstoffmonoxid. Die CO-Freisetzung wurde auch mit Hilfe eines des fluoreszenten CO-Indikators COP-1 untersucht. Während bei Inkubation im Dunkeln auch bei einem 10:1- Verhältnis von CORM zu COP-1 kein Signal beobachtet werden konnte, führt die lichtinduzierte CO-Freisetzung zu einem konzentrationsabhängigen Anstieg der Fluoreszenz. Die photoaktivierte CO-Freisetzung wurde auch in lebenden HUVEC- Zellen untersucht. In der überstehenden Lösung konnte ein Ansteig der COP-1- Fluoreszenz relativ zum Hintergrund um einen Faktor von 15 beobachtet werden während die Intensititäszunahme für die Zellfraktion nur bei etwa 5-fach lag. Dies könnte auf eine nur geringe Zellaufnahme von COP-1 oder CORM oder beider Moleküle zurückzuführen sein. Die Untersuchungen etablieren die bpea-Komplexe aber eindeutig als photoaktivierbare CO-releasing molecules (PhotoCORMs). Peptide sind attraktive Trägersysteme für das cellular delivery von Metallkomplexen. Als Modell für solche Trägerpeptide wurde die transforming growth factor -bindende (TGF-) Sequenz durch Festphase-Peptidsynthese hergestellt. Die Anknüpfung von [Mn(bpea)(CO)3]+ an das Peptid über eine periphere Funktionalität des Liganden über eine Sonogashira-Kreuzkupplung bzw. CuAAC-"Click"-Reaktion sollte in einer Postlabelling-Strategie erfolgen. Obwohl verschiedene Bedingungen getestet wurden führte erstere Reaktion jedoch nicht zum Erfolg. Die CuAAC-Reaktion zwischen einem Alkin-funktionalisierten Metallkomplex und einem Azid-terminierten Peptid führt dagegen zu dem gewünschten N-terminal funktionalisierten Konstrukt, welches über eine Triazolgruppe zusammengehalten wird. Trotzdem zeigte das Konjugat auf Grund einer Imin-Bindung im bpea-Liganden eine Tendenz zur Hydrolyse in wässrigem Medium. Als Alternative wurde daher die milde und katalysatorfreie Oxim-Ligation in Verbindung mit einem stabileren, Amin- anstatt Imin-basierten Liganden untersucht. Die Kupplung zwischen einem Aminoxyessigsäure- terminierten TGF--bindenden Peptid und einem Aldehyde-funktionalisierten Metallkomplex ergab das gewünschte Konjugat in guter Ausbeute mit höher Stabilität. Für bis zu 96 h konnte selbst bei wiederholten freeze-thaw-Zyklen keinerlei Zersetzung beobachtet werden. Das CO-Freisetzungsverhalten von Konjugat und Stammverbindung war identisch, die Photoaktivierung bei 365 nm führt für beide innerhalb von 1–1.5 h zur Freisetzung von zwei Äquivalenten CO pro Mol Komplex. Die Oxim-Ligation konnte so als milder Zugang zu CORM-Peptidkonjugaten etabliert werden. Dendrimere sind Baum-artige Moleküle mit einer Vielzahl von Funktionalitäten in der Peripherie, die eine Modifikation mit Metallkomplexen für biologische Anwendungen erlauben. Von besonderem Interesse ist hierbei die Anreicherung in Tumorgewebe auf Grund des enhanced permeability and retention(EPR)-Effekts. Diaminobutan(DAB)- und Polyamidoamin(PAMAM)-Dendrimere der Generation 1 mit vier terminalen Amingruppen wurden daher in einer Schiff-Base-Kondensation mit Aldehyd-funktionalisierten Mn(bpea)(CO)3-Komplexen umgesetzt. Die erhaltenten Metallkomplex-Dendrimer-Konjugate waren in wässriger DMSO-Lösung im Dunkeln für bis zu 14 h stabil. Die Photolyse zeigte eine geringfügig schnellere CO- Freisetzung für das DAB- vs. dem PAMAM-Dendrimer. Eine Anregung bei 410 nm führte zu einer deutlich langsameren CO-Freisetzung gegenüber der 365 nm- Belichtung. Mit Hilfe des Myoglobin-Assays konnte gezeigt werden daß auf diese Weise 50–55% der Gesamtzahl an CO-Liganden aus dem System freigesetzt werden kann. Im Rahmen der vorliegenden Arbeit wurden neue Mangan(I)tricarbonyl-Komplexe auf der Basis des tridentaten Bis(pyrazolyl)ethylamin(bpea)-Liganden hergestellt, die in der Peripherie funktionalisierte Phenylgruppen tragen, welche die Anknüpfung an biologische Trägersysteme auf der Basis von Peptiden und Dendrimeren erlauben. Auf Grund ihrer Stabilität unter Lichtausschluß sind diese Verbindungen als CORM- Prodrugs geeignet. Die Photoaktivierung bei 365 nm führt zur Freisetzung von zwei Äquivalenten CO pro Mol CORM, wobei die Stammverbindungen eine etwas schnellere Kinetik aufweisen als die Konjugate. Insbesondere die Dendrimer- basierten Systeme können auf Grund der hohen Anzahl von Mn(CO)3-Gruppen bis zu sieben CO pro Mol Konjugat liefern. Für die Zukunft bleibt zu zeigen ob diese Konjugate eine zelluläre Anreicherung für biologische Anwendungen erlauben wird

碩士
高雄醫學大學
醫藥暨應用化學研究所
97
One of the important features for drug delivery is drug carrier. Hitherto, there are a variety of drug carriers, including ionic polymer, micelle and dendrimers. Drug carrier not only achieves the purpose of targeting delivery, but also increase volume of drug, so that prolong the duration by diminish the secretion from kidney. Unfortunately, the ability to release the carried drug at specific site is not well-studied until now. The objective of this thesis is to design a novel drug carrier for specific release. Accordingly, prostate specific antigen (PSA) was selected as platform to demonstrate this principle and a specific peptide with sequence of SSYYSG, which is proved as ligand of PSA, was introduced to the surface of dendrimer. While the presence of PSA, the dendrimers functionalized with GRLSSYYSGCGGG will be hydrolyzed and causes the size of dendrimers shrinks. We postulate the carrying drug will be free due to the decreasing of volume. Despite of the applying of solid-phase-peptide-synthesis, the preparation and purification of long peptides remains challenge. To avoid the tedious purification process, ligation strategy was applied to reduce the synthetic effort. We have developed a reliable protocol for manually solid phase peptide synthesis, and the desired peptides GRLSSYYSG and CGGG has been prepared and identified by NMR and mass spectrum. Regarding to the drug release experiment, we modify the dendrimer surface by peptide sequence for found the relationship between the volume of drug carrier and the amount of encapsulated. We have coupled G:2-4 PAMAM dendrimers with the GRLSSYYSG peptide, meanwhile, compared the volume of encapsulated with commercial PAMAM dendrimer.

Dissertação de mestrado em Biofísica em Bionanossistemas
Todas as células, unidade de vida, apresentam-se compartimentalizadas por uma bicamada lipídica permitindo que numerosos processos ocorram á superfície dos organelos por uma associação reversível de proteínas do citosol. Nestes processos simples parâmetros físicos como, curvatura membranar e/ou densidade de carga poderão ser importantes “inputs” para criar respostas temporais e espaciais.(Bigay & Antonny, 2012). A maioria dos estudos encontram-se focados em parâmetros físico-quimicos das biomoléculas como os maiores reguladores na interacção com membranas. Deste modo o papel desempenhado pelas propriedades membranares não se encontram completamente elucidados. Neste projecto, eu irei investigar a um nível singular as propriedades físicas das biomembranas e a sua directa influência na associação reversível com biomoléculas. Para este efeito, eu irei utilizar biomoléculas com função definida e analisar o seu comportamento quando importantes parâmetros associados as mesmas são manipulados. De modo a obter estes objectivos, irei utilizar um ensaio “in-vitro”, onde lipossomas fluorescentes individuais serão imobilizados numa superfície de vidro e visualizados usando microscopia laser confocal. Deste modo, eu planeei o uso de lipossomas singulares e monitorizar como os parâmetros membranares afectam a ligação de três classes diferentes de moléculas: primeiramente um péptido lipidado derivado da GTPase N-Ras (tN-Ras), para o estudo da forma da membrana na ligação peptídica. Em segundo o dendrimero (PAMAM G6) para elucidar o papel desempenhado pelas interacções hidrofóbicas e electroestáticas. Por último um péptido derivado da proteína membranar myristoyled alanine-rich C kinase substrate (MARCKS), para o estudo de como o pH de lipossomas individuais poderão regular o recrutamento específico de péptidos altamente catiónicos.
All cells, the unit of life, are compartmentalized by a lipid bilayer allowing numerous molecular processes to occur at the surface of organelles through reversible association of proteins from the cytosol. In these processes simple physical parameters such as membrane curvature and/or charge density can be important inputs to create sharp temporal and spatial responses.(Bigay & Antonny, 2012) Most studies focus on the physicochemical parameters of the biomolecules as the major regulators of the interaction with membranes. This means that the role played by membrane properties are still not fully understood. In this project, I will investigate at single level the physical properties of biomembranes and their direct influence in the reversible association with biomolecules. For this, I will be using biomolecules with defined function to analyze the behavior when important parameters associated with biomembranes are manipulated. To accomplish these goals, I will use an in vitro assay, where individual fluorescent liposomes are immobilized on a glass surface and imaged using confocal microscopy. Thus, I planned use of single liposomes and monitor parameters affecting the membrane binding of three different classes of molecules: first lipidated peptide derived from the NGTPase RAS (tN-RAS) for the study of the membrane shape in the peptide bond. Secondly, a dendrimer (PAMAM G6), to elucidate the role of hydrophobic and electrostatic interactions. Finally, a peptide derived from the membrane protein myristoyled alanine-rich C kinase substrate (MARCKS), to study how the individual pH of liposomes may regulate the recruitment of specific highly cationic peptide.

In this study, cyclic arginine-glycine-aspartic acid (RGD) peptide-modified amine-terminated generation 5 poly(amidoamine) (G5.NH2 PAMAM) dendrimers were prepared for the encapsulation of the anticancer drug doxorubicin (DOX) for targeted delivery to cancer cells overexpressing αvβ3 integrin cell surface receptors. First, the thiolated RGD peptide was linked to polyethylene glycol (PEG) via the bifunctional cross-linking reagent 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS). Then a dendrimer modification process was performed in which the PEGylated RGD peptide and fluorescein isothiocyanate (FI) were covalently attached to the G5 dendrimers. This process was finally followed by acetylation of the remaining dendrimer terminal amines. The experimental results show that each G5.NHAc-FI-PEG-RGD dendrimer approximately encapsulated six DOX molecules. This formed complex is water soluble and stable. In vitro release studies proved that the multifunctional dendrimers facilitate a sustained release of DOX. More interesting, one-dimensional NMR and two-dimensional NMR were applied to investigate the interactions between dendrimers and DOX. Here, the impact of the environmental pH on the release rate of DOX from G5.NHAc-FI-PEG-RGD/DOX was fully studied. Furthermore, cell biological studies demonstrated that G5.NHAc-FI-PEG-RGD dendrimers have no cytotoxicity towards U87-MG cancer cells but that G5.NHAc-FI-PEG-RGD/DOX complexes have almost the same cytotoxicity as DOX alone. Moreover, due to the targeting ability of RGD, this dendrimer/drug system can also specifically target and display therapeutic efficacy to cancer cells overexpressing αvβ3 integrins. The cellular internalization of the multifunctionalized dendrimer was shown to be receptor mediated to an important extent. According to this study, we can say that G5.NHAc-FI-PEG-RGD is a promising system for the targeted therapy of different types of cancer.
Neste trabalho, foram preparados dendrímeros de poli(amidoamina) (PAMAM) de geração 5 (G5) funcionalizados com o péptido cíclico RGD para o encapsulamento do fármaco anticancerígeno doxorubicina (DOX) e sua entrega em células cancerígenas que expressem elevadas quantidades de integrinas αvβ3 na sua superfície (entrega específica do fármaco em células-alvo). No processo de síntese, o péptido contendo um grupo tiol foi primeiro ligado a uma cadeia de polietilenoglicol (PEG) através de um reagente de reticulação bi-funcional. De seguida, os dendrímeros foram ligados covalentemente ao péptido PEGilado e, ainda, ao isotiocianato de fluoresceína (FI), seguindo-se a acetilação (Ac) das aminas terminais remanescentes no dendrímero para se obter o sistema final G5.NHAc-FI-PEG-RGD. Os resultados experimentais mostram que, aproximadamente, existem 6 moléculas de DOX encapsuladas por G5.NHAc-FI-PEG-RGD, sendo estes complexos solúveis e estáveis em água. Os estudos in vitro mostraram que a libertação do fármaco a partir dos dendrímeros multifuncionalizados é controlada. O trabalho envolveu, ainda, estudos de NMR mono- e bi-dimensional na investigação da interacção existente entre os dendrímeros e as moléculas de DOX, e ainda a avaliação do impacto do pH ambiental na velocidade de libertação da DOX. Realizaram-se, igualmente, estudos biológicos com células U87-MG, os quais mostraram que os sistemas G5.NHAc-FI-PEG-RGD não apresentavam toxicidade e que, quando complexados com a DOX, apresentavam uma citotoxicidade semelhante à do fármaco usado de forma isolada. Dada a afinidade do péptido RGD para as integrinas presentes em grande quantidade à superfície das células U87-MG, o sistema G5.NHAc-FI-PEG-RGD mostrou-se muito eficaz na entrega específica do fármaco e consequente eficácia terapêutica. A entrega do fármaco nas células mostrou ser, numa importante extensão, mediada pelos receptores (integrinas αvβ3) presentes à sua superfície. Este trabalho mostrou que os dendrímeros multifuncionalizados G5.NHAc-FI-PEG-RGD são RESUMO     vi bastante promissores como sistemas para a entrega específica de fármacos em células cancerígenas.

Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células Estaminais
Polyphenols are extensively studied due to their wide range of bioactivities. In this thesis we used this knowledge to evaluate a series of cork-based polyphenols/extracts to act: as anti-bacterial agents (Chapter 3); in the reduction of UV-mediated toxicity (Chapter 4); and as modulators of amyloid-b (Ab) aggregation/cytotoxicity (hallmark of Alzheimer’s disease, AD, Chapter 5). We found that vescalagin/castalagin (extracted/purified from cork powder) are the main responsibles for the bioactivities of cork extracts due to their galloyl (Ga) units. Cork extracts rich in vescalagin/castalagin protected cells from UV-mediated cell death (Chapter 4). They also inhibited the folding of amyloid-like peptides/proteins from the extracellular matrix of bacteria, blocking the formation of biofilms (Chapter 3); as well as to remodel the Ab aggregation into non-toxic forms (Chapter 5). We then synthesized (Chapter 6) three Gapresenting dendrimers that recapitulate the vescalagin/castalagin bioactivities, and showed that dendrimers with 6 Ga units present a compromise between bioactivity and hydrophilicity enabling their use in the cellular environment while drastically reducing the presence of toxic Ab oligomers. We also showed (Chapter 7) that glycosaminoglycans’ (GAGs) building blocks and their analogues, e.g. glucosamine-6-sulfate (GlcN6S) and glucosamine-6-phosphate (GlcN6P), complement the bioactivities of vescalagin/castalagin in the context of AD by: modulating the Ab-mediated toxicity; reducing membrane dysfunction; re-establishing healthy insulin signaling; lowering the acetylcholinesterase activity to basal levels; and reducing inflammation markers. We further developed a self-assemble monolayer (SAM) platform (Chapter 8) able to recapitulate the influence of the GAGs sulfation degree on the presentation of fibronectin (Fn) and its impact in the adhesion/morphology of stem cells. Overall, we show that corkbased compounds, e.g. vescalagin/castalagin, present a wide range of bioactivities (e.g. anti-UV, antibacterial, anti-amyloidogenic) that are recapitulated by Ga-presenting dendrimers. Vescalagin/castalagin/Ga-terminated dendrimers can be used alone or in combination with GlcN6S or GlcN6P to reduce AD hallmarks. We also show that the sulfation degree presented on SAM-coated surfaces is critical for their bioactive character.
Os polifenóis são muito estudados dado a sua ampla bioatividade. Nesta tese, usámos este conhecimento para avaliar vários compostos/extratos da cortiça para atuarem como: anti-bacterianos (Capítulo 3); anti- UV (Capítulo 4); e moduladores da agregação/toxicidade da b-amilóide (Ab) (associado à doença de Alzheimer, AD, Capítulo 5). Demonstrámos que a vescalagina/castalagina presentes nos extratos de cortiça são essenciais para a sua bioatividade, dada a presença do grupo galoíl (Ga). Extratos ricos em vescalagina/castalagina reduziram a morte celular mediada pela radiação UV (Capítulo 4) e inibiram a organização dos péptidos/proteínas da matriz extracelular de bactérias, bloqueando a formação de biofilme (Capítulo 3). A vescalagina/castalagina foram ainda capazes de remodelar o Ab em formas não tóxicas (Capítulo 5). Também sintetizámos dendrímeros apresentando a unidade Ga (Capítulo 6) mimetizando a bioatividade da vescalagina/castalagina. O dendrímero com 6 Gas apresentou um compromisso entre bioatividade/hidrofilicidade, permitindo a sua utilização no ambiente celular e reduzindo a toxicidade dos oligómeros de Ab. Provámos ainda (Capítulo 7) que a glucosamina-6-sulfato (GlcN6S) e a glucosamina-6-fosfato (GlcN6P) apresentam bioatividade complementar à da vescalagina/castalagina no contexto da AD, reduzindo a toxicidade do Ab, protegendo a membrana celular, normalizando a sinalização da insulina, reduzindo a atividade da acetilcolinesterase e a expressão dos marcadores de inflamação. Também desenvolvemos uma plataforma de “self-assemble monolayers” (SAMs, Capítulo 8) capaz de mimetizar a influência do grau de sulfatação dos glicosaminoglicanos (GAGs) na apresentação de fibronectina e o seu impacto na adesão/morfologia de células estaminais. Em resumo, identificámos uma vasta bioatividade da vescalagina/castalagina (ex. anti-UV, anti-bacteriana, anti-amiloidogénica) que pode ser mimetizada por dendrímeros apresentando unidades de Ga. A vescalagina/castalagina/dendrímeros de Ga são capazes de reduzir as várias características da AD, de uma forma individual ou em combinação com GlcN6S/GlcN6P. Também demonstrámos que o grau sulfatação apresentado em superfícies cobertas por SAMs é critico para o seu carácter bioativo.
I would like to acknowledge the funding, specifically the Programa Operacional Regional do Norte (Norte 2020): NORTE-08-5369-FSE-000037 (PhD scholarship) and NORTE-01-0246-FEDER-000022 (Northern Discoveries: The Discoveries CTR – Setting Sails). This work was also supported by the European Commission: FP7-REGPOT-CT2012-316331-POLARIS, H2020-TWINN-2015-692333– CHEM2NATURE, 739572-THE DISCOVERIES CTR and the Portuguese Foundation for Science and Technology (FCT): AL ICVS/3Bs #50026.