A funcionalização de superfície nas nanocápsulas contendo doxorrubicina com o peptídeo RGD é uma estratégia promissora devido a ligação preferencial na integrina αvβ3 expressa em células tumorais. Este estudo objetivou o desenvolvimento, caracterização e estudos biológicos de nanocápsulas multiparedes com doxorrubicina e funcionalizadas com RGD. Para isso, na primeira etapa do trabalho foi realizada a síntese do peptídeo RGD. Os produtos obtidos foram caracterizados por análises de infravermelho e RMN de 1H. Na segunda etapa foram desenvolvidas formulações de nanocápsulas com doxorrubicina ou cloridrato de doxorrubicina, e, nanocápsulas multiparedes revestidas com quitosana, íons zinco, RGD ou fenilalanina. Essas suspensões foram caracterizadas através da determinação do pH, diâmetro de partícula por diferentes técnicas, potencial zeta, eficiência de encapsulação e eficiência de associação do RGD na superfície da nanopartícula. Na terceira etapa, foram realizados ensaios de viabilidade celular por MTT após 24 e 72h com as formulações desenvolvidas em células de câncer de mama (MCF7) e glioblastoma humano (U87MG). As formulações apresentaram diferentes valores de citotoxicidade e, utilizando o Gráfico de Pareto foi possível determinar os fatores que exercem maior influencia. Em células MCF7 foi a concentração de fármaco e tempo de tratamento e, nas células U87MG além desses fatores, a funcionalização mostrou-se determinante. Além disso, foi avaliada a captação das nanocápsulas funcionalizadas com RGD e fenilalanina após 24h nas células tumorais e células de queratinócitos humanos (HaCat), com diferentes níveis de expressão da integrina αvβ3. O estudo mostrou menores valores de captação nas células HaCat (sem expressão de integrina αvβ3) para as duas formulações testadas. Finalmente as nanocápsulas funcionalizadas com RGD apresentaram maior captação em células U87MG com maior expressão da integrina αvβ3. / The surface functionalization in nanocapsules containing doxorubicin with RGD peptide is a promising strategy due to preferential binding in the αvβ3 integrin expressed on tumor cells. This study aimed the development, characterization, and biological studies of multiwall nanocapsules containing doxorubicin and functionalized with RGD. For this reason, in the first stage of this study the synthesis of RGD peptide was performed and the products characterized by infrared analysis and 1H NMR. Besides, nanocapsules formulations were developed containing doxorubicin or doxorubicin hydrochloride, and multiwall nanocapsules coated with chitosan, zinc ions, RGD or phenylalanine. These suspensions were characterized by pH determination, particle diameter by different techniques, zeta potential, encapsulation efficiency, and association efficiency of RGD on the surface of the nanoparticle. Additionally, it was performed cell viability assays by MTT after 24 and 72 hours with formulations developed in breast cancer (MCF7) and human glioblastoma cells (U87MG). Formulations showed different cytotoxicity values. The Pareto chart was possible to determine factors that have more influence. In MCF7 cells was drug concentration and treatment time, and U87MG cells, besides these factors, the functionalization was decisive. Furthermore, it was performed the cellular uptake of nanocapsules functionalized with RGD or phenylalanine after 24 hours in tumor cells and human keratinocyte cells (HaCaT), with different levels of expression αvβ3 integrin. The study showed less uptake in HaCaT cells (without expression αvβ3 integrin) for the two formulations applied, and the nanocapsules functionalized with RGD showed more uptake in U87MG cells, with higher expression of integrin αvβ3.

Ageing is a natural part of every human life cycle. During ageing there are lots of changes in the organism. One of the main pillars of the cosmetics industry is the development of active compounds that are fighting signs of skin ageing. The components as bioactive peptides are considered to be promising anti-ageing products, mainly because of the possibility to precisely define their chemical structure and therefore achieve more effective biological targeting. One of the signs of skin ageing is the weakening of the connections between epidermal cells and the extracellular matrix, decreasing expression of adhesion molecules and molecular components of dermo-epidermal connection. The most abundant adhesive receptors in the skin are integrins. Their ligands are extracellular matrix molecules, e. g. laminin or fibronectin. The minimal recognition sequence of integrins receptors is the amino acid motif arginine- glycine- aspartic acid (RGD). This sequence is also very interesting in terms of cosmetic applications because it provides the ability to create new and effective bioactive peptides. The subject of present work is basic safety testing of four peptides with RGD motif. A sequence of three peptides was modified by addition of glycine amino acids, or alanine. The expected effect was to improve interaction with integrin receptors. In each of the sample, levels of endotoxin was determined in order to exclude any possible interfering effects on the viability of cells. Subsequently, in NIH3T3 mouse fibroblasts viability was monitored by MTT assay and morphology. The quantity of obtained protein had been determined to increase data interpretation relevance.

Diagnóstico precoce e tratamento de melanoma, um tumor cutâneo com o pior prognóstico, é extremamente importante para um resultado clínico favorável. A biblioteca de peptídeos phage display é um recurso útil de triagem para identificar peptídeos bioativos que interagem com alvos em cânceres. O objetivo desse estudo foi a avaliação de dois traçadores de tecnécio-99m com sequências peptídicas RGD e NGR, conjugados com o quelante bifuncional MAG3. Os conjugados peptídicos (10 μL de uma solução μg/μL) foram marcados com tecnécio-99m usando tampão de tartarato de sódio. A avaliação radioquímica foi feita por ITLC e confirmada por CLAE. O coeficiente de partição foi determinado e ensaios de internalização foram realizados em duas linhagens celulares de melanoma (B16F10 e SKMEL28). A avaliação da biodistribuição dos traçadores foi realizada em animais sadios em diferentes tempos e também em camundongos portadores de células tumorais aos 120 min após a sua administração. Estudos de bloqueamento também foram conduzidos pela co-injeção de peptídeo frio. O desempenho dos conjugados peptídicos mostraram-se bastante parecidos em diversas avaliações. Eles foram radiomarcados com alta pureza radioquímica (>97%). Ambos são hidrofílicos, com excreção renal preferencial. A captação tumoral foi maior para células SKMEL28 do que para as células B16F10, especialmente para o 99mTc-MAG3-PEG8-c(RGDyK) (7,85±2,34 %DI/g) aos 120 min pós-injeção. O desempenho do 99mTc-MAG3-PEG8-c(RGDyk) foi superior que o do traçador com NGR, quanto à captação no melanoma humano podendo ser considerado como um promissor radiofármaco para diagnóstico de melanoma. / Early diagnosis and treatment of melanoma, a cutaneous tumor with a serious prognosis, is extremely important for optimal clinical outcome. Phage display peptide libraries are a useful screening resource for identifying bioactive peptides that interact with cancer targets. The aim of this study was the evaluation of two technetium-99m tracers for angiogenesis detection in melanoma model, using cyclic peguilated pentapeptide with RGD and NGR motifs conjugated with bifunctional chelator MAG3. The conjugated peptides (10 μL of a μg/μL solution) were labeled with technetium-99m using a sodium tartrate buffer. Radiochemical evaluation was done by ITLC and confirmed by HPLC. Partition coefficient was determined and internalization assays were performed in two melanoma cells (B16F10 and SKMEL28). Biodistribution evaluation of the tracers was done in healthy animals at different times and also in mice bearing the tumor cells at 120 min post injection. Blocking studies were also conducted by co-injection of cold peptides. The conjugated showed the same profile in many evaluations. They were radiolabeled with high radiochemical purity (>97%). Both were hydrophilic, with preferential renal excretion. Tumor uptake was higher for human melanoma cells than for murinic melanoma cells, specially for 99mTc-MAG3-PEG8-c(RGDyK) (7.85±2.34 %ID/g) at 120 min post injection. The performance of 99mTc-MAG3-PEG8-c(RGDyk) was much better than NGR tracer concerning human melanoma uptake and might be considered in future investigations focusing radiotracers for melanoma diagnosis.

Diagnóstico precoce e tratamento de melanoma, um tumor cutâneo com o pior prognóstico, é extremamente importante para um resultado clínico favorável. A biblioteca de peptídeos phage display é um recurso útil de triagem para identificar peptídeos bioativos que interagem com alvos em cânceres. O objetivo desse estudo foi a avaliação de dois traçadores de tecnécio-99m com sequências peptídicas RGD e NGR, conjugados com o quelante bifuncional MAG3. Os conjugados peptídicos (10 μL de uma solução μg/μL) foram marcados com tecnécio-99m usando tampão de tartarato de sódio. A avaliação radioquímica foi feita por ITLC e confirmada por CLAE. O coeficiente de partição foi determinado e ensaios de internalização foram realizados em duas linhagens celulares de melanoma (B16F10 e SKMEL28). A avaliação da biodistribuição dos traçadores foi realizada em animais sadios em diferentes tempos e também em camundongos portadores de células tumorais aos 120 min após a sua administração. Estudos de bloqueamento também foram conduzidos pela co-injeção de peptídeo frio. O desempenho dos conjugados peptídicos mostraram-se bastante parecidos em diversas avaliações. Eles foram radiomarcados com alta pureza radioquímica (>97%). Ambos são hidrofílicos, com excreção renal preferencial. A captação tumoral foi maior para células SKMEL28 do que para as células B16F10, especialmente para o 99mTc-MAG3-PEG8-c(RGDyK) (7,85±2,34 %DI/g) aos 120 min pós-injeção. O desempenho do 99mTc-MAG3-PEG8-c(RGDyk) foi superior que o do traçador com NGR, quanto à captação no melanoma humano podendo ser considerado como um promissor radiofármaco para diagnóstico de melanoma. / Early diagnosis and treatment of melanoma, a cutaneous tumor with a serious prognosis, is extremely important for optimal clinical outcome. Phage display peptide libraries are a useful screening resource for identifying bioactive peptides that interact with cancer targets. The aim of this study was the evaluation of two technetium-99m tracers for angiogenesis detection in melanoma model, using cyclic peguilated pentapeptide with RGD and NGR motifs conjugated with bifunctional chelator MAG3. The conjugated peptides (10 μL of a μg/μL solution) were labeled with technetium-99m using a sodium tartrate buffer. Radiochemical evaluation was done by ITLC and confirmed by HPLC. Partition coefficient was determined and internalization assays were performed in two melanoma cells (B16F10 and SKMEL28). Biodistribution evaluation of the tracers was done in healthy animals at different times and also in mice bearing the tumor cells at 120 min post injection. Blocking studies were also conducted by co-injection of cold peptides. The conjugated showed the same profile in many evaluations. They were radiolabeled with high radiochemical purity (>97%). Both were hydrophilic, with preferential renal excretion. Tumor uptake was higher for human melanoma cells than for murinic melanoma cells, specially for 99mTc-MAG3-PEG8-c(RGDyK) (7.85±2.34 %ID/g) at 120 min post injection. The performance of 99mTc-MAG3-PEG8-c(RGDyk) was much better than NGR tracer concerning human melanoma uptake and might be considered in future investigations focusing radiotracers for melanoma diagnosis.

L’objectif du travail présenté dans cette thèse était de développer des nanoparticulesfonctionnelles ayant la capacité d’induire l’adhésion et la migration de kératinocyteshumains normaux. L’utilisation de systèmes particulaires pour favoriser l’adhésion etla migration cellulaire dans les processus de cicatrisation constitue une nouvelleapproche de l’ingéniérie tissulaire. Dans cette optique, un dérivé hydrosoluble du chitosan rendu fonctionnel par l’ajoutde peptides RGD a été développé. Les nanoparticules furent développées parcoacervation complexe entre le dérivé cationique du chitosan et le sulfate dechondroïtine anionique. La capacité du système particulaire à induire unchangement cellulaire phénotypique a été évaluée in vitro.Lors de l’évaluation de ce nouveau polymère, le succès de la synthèse a été montrépar l’absence de cytotoxicité et par la préservation de son activité biologique médiéepar les séquences RGD. Aussi bien les polymères que les nanoparticules ont induitl’adhésion et la mobilité de fibroblastes dermiques humains, confirmant le conceptde nanoparticules bio-actives. Cependant, concernant l’étude des interactions entreles nanoparticules et les kératinocytes, aucune conclusion n’a pu être tirée etd’autres travaux sont nécessaires. Pour résumer, un système particulaire bio-actif a été développé. Le choix despeptides RGD pour induire la migration des kératinocytes doit être réévalué, etl’utilisation de concentrations plus importantes, de mélange de peptides d’adhésionou l’utilisation de peptides d’adhésion différents doit être envisagée pour laréalisation d’études ultérieures. / The aim of the work presented in this thesis, was to develop functionalizednanoparticles with the ability to induce adhesion and migration in normal humankeratinocytes. Using particulate systems to promote and support cell adhesion andmigration in epidermal restoration is a novel approach of tissue engineering.In this view, a water-soluble chitosan derivative functionalized with RGD peptideswas developed. Nanoparticles were formed through complex coacervation betweenthe cationic chitosan derivative and the anionic chondroitin sulfate. The particulatesystem was evaluated in vitro for its ability to change phenotype in cells.In the evaluation of the novel hybrid polymer, the successful synthesis wasconfirmed by the absence of cytotoxicity and a preserved bioactivity specific to theRGD-moieties. Both the polymer and the particles formed thereof induced celladhesion and spreading in human dermal fibroblasts, proving the concept ofbioactive nanoparticles. However, when investigating the interaction between thenanoparticles and keratinocytes, no clear conclusion could be drawn and furtherassays are required. To summarize, a bioactive particulate system was developed. The choice of RGDpeptides to induce migration in keratinocytes needs to be re-evaluated and higherconcentrations, mixtures of adhesion peptides or other adhesion peptides might beconsidered for further investigations.

Le ciblage de la néoangiogenèse tumorale est une voie actuelle de recherche pour le diagnostic et pour le traitement des tumeurs solides. Les cellules endothéliales des néovaisseaux tumoraux surexpriment certains marqueurs spécifiques tels que l'intégrine avb3, qui reconnaît spécifiquement les peptides possédant le motif « RGD » (-Arg-Gly Asp-). Nous avons étudié le potentiel d'un nouveau radiotraceur - le RAFT-RGD - en vue d'imagerie moléculaire nucléaire des néovaisseaux. In vitro, le couplage de 4 c(RGDfK) au RAFT augmente la captation du traceur par les cellules avb3 positives, par rapport au c(RGDfK). De plus, le RAFT-RGD possède une meilleure affinité que le c(RGDfK) et des propriétés d'inhibition de l'angiogenèse similaires. In vivo, l'ensemble des tumeurs, avb3 positives et négatives, est visible par imagerie planaire non invasive corps entier, comme en SPECT, dès 30 min post-injection et au-delà de 24 h, grâce à une gamma caméra dédiée au petit animal. Le RAFT-RGD, malgré l'absence d'amélioration du contraste par rapport au cRGD, semble être un traceur prometteur de l'angiogenèse tumorale : il pourrait renseigner sur l'évolution de la pathologie et le suivi de l'efficacité des traitements des tumeurs dans les services de<br />Médecine Nucléaire. De plus, par sa structure chimique, le RAFT-RGD apporte de multiples possibilités de marquage (émetteurs γ et β-), ce qui permet d'envisager des applications intéressantes notamment dans le domaine thérapeutique (radiothérapie interne vectorisée).

München, Techn. Univ., Diss., 2003.

München, Techn. Univ., Diss., 2003.

Universiẗat, Diss., 2001--München.

La première partie de cette thèse est dédiée à la modification de polyélectrolytes pour former des films de multicouche de polyélectrolytes (PEM) ayant des propriétés d’adhésion de protéine et de cellules bien contrôlées et modifiables par étirement. L’acide polyacrylique a été modifié avec des groupes latéraux phosphorylcholine (PC) à des taux de 25 % (PAA-PC) ou avec des chaînes oligo(éthylène oxyde) terminées par la biotine : (EO)nBiotine (n = 0, 3, 9 et 18) avec de taux de modification de 1, 5, 10 ou 25 %. Des PEM incorporant ces polymères lient spécifiquement la streptavidine et repoussent tout autre protéine. Les propriétés d’adsorption et la sélectivité de ces PEM ont été mesurées par microbalance à quartz. Sur un substrat de PDMS étirables, on a construit des PEM terminés par un PAA portant des RGD recouvert par deux couches contenant PAA-PC. Au repos, seuls les PC sont exposés et inhibent l’adhésion cellulaire ; sous étirement, les groupes RGD sous-jacents sont exposés et déclenchent l’adhésion de fibroblastes.La deuxième partie est consacrée à l‘étude d’acide polyméthacrylique modifié hydrophobiquement avec des chaînes alkyle liées par des esters à la chaîne principale. 3 chaînes différentes ont été greffées : -C12H25 ; -C18H35 et C4H8-OOC- C11H23 avec des taux de 1, 5 and 10 %. Ces polymères sont associatifs et forment des hydrogels dans des tampons physiologiques pour des taux de modifications de 5% et des concentrations supérieures à 4% en poids. Ces gels ont été caractérisés par des mesures rhéologiques. Leur incubation avec des lipases provoque une baisse de leur viscosité, interprétable par une coupure des esters. Quand les gels faits à partir du PAA-C12 sont incubés avec une culture de Pseudomonas aeruginosa, la viscosité baisse également, ce qui montre que les chaînes sont également coupées in vivo. / The first part of this thesis is dedicated to the modification of polyelectrolytes to form polyelectrolyte films with controlled and stretch responsive cell and protein adsorption properties. Poly(acrylic acid) (PAA) was modified with side phosphorylcholine groups (PC) at rates of 25 % or with oligo(ethylene oxide) chains ended by biotin ((EO)nBiotin, (n =0, 3, 9 and 18) at 1, 5, 10 and 25 % modification rates. Polyelectrolytes multilayer films (PEM) containing these polyelectrolytes bind selectively streptavidin but repel all other proteins. The adsorption properties and selectivity were measured by quartz crystal microbalance. On a stretchable PDMS substrate, we have built PEM ended by PAA bearing RGD, covered by two PAA-PC layers on the top. Under rest, only the PC groups are exposed and prevent cell adhesion; when the film is stretched, the underlying RGD groups are exposed, and trigger adhesion of fibroblasts.The second part was consecrated to the study of poly(methacrylic acid) hydrophobically modified with alkyl chains connected through an ester moiety to the main chain. Three different chains were grafted -C12H25; -C18H35 and -C4H8- OOC-C11H23 with a rate of 1, 5 and 10 %. These polymers associate in water and form hydrogels in physiological buffer, for modification rates higher than 5 % and polymer concentrations higher than 4 wt. %. The gels were characterized by rheology. Their incubation with lipases resulted in a decrease of their viscosity, which could be interpreted by the cleavage of the hydrophobic side chains, by rheological tests. When the gels with PAA-C12 were incubated with a culture of Pseudomonas aeruginosa, their viscosity decreased, which shows that alkyle chains are also cleaved in vivo.