La thérapie photodynamique (PDT) est un type de traitement du cancer qui offre de nombreux avantages. Une stratégie pour améliorer l'efficacité de la PDT est l'élaboration de photosensibilisateurs (PSs) de troisième génération, composés d’une molécule photoactivable couplée à un agent de ciblage. Les travaux de recherche de cette thèse portent sur l'amélioration de la sélectivité du traitement PDT en concevant des PSs ciblés. La première partie de la thèse est consacrée à l'étude d'un PS couplé à de l’acide folique (PS-FA). L’acide folique est une unité bien connue de ciblage qui se lie de façon efficace au récepteur de l'acide folique sur-exprimé à la surface de nombreuses cellules cancéreuses. Nous avons particulièrement vérifié la stabilité de l'acide folique sous l'influence des facteurs environnementaux. La deuxième partie est consacrée à l'étude du peptide KDKPPR conçu pour cibler neuropiline-1, un récepteur surexprimé dans les néovaisseaux. Plusieurs modifications du peptide ont été faites et les analogues ont été testés par des analyses ELISA afin d'évaluer leur capacité de liaison à la suite des modifications. La troisième partie de la thèse a consisté en la synthèse de porphyrines couplées à des blocs porteurs de 1, 2 ou 3 peptides grâce à la technique de chimie click pour former de multiples conjugués avec des nombres différents de porphyrines et peptides attachés aux plateformes / Photodynamic therapy (PDT) is a type of cancer therapy that could offer many advantages. One possible way to improve the effectiveness of PDT is the elaboration of third generation photosensitizers (PSs) which consisted of PSs coupled with targeting agents. This thesis focuses on improving the selectivity of PS delivery through designing targeted PS agents. The first part of the thesis deals with the study of a PS-folic acid (PS-FA). FA is a well-known targeting unit which bonds with high efficiency to folic acid receptor over-expressed on the surface of many cancer cells. We particularly checked the stability of folic acid under the influence of environmental factors. The second part is devoted to the study of KDKPPR peptide designed to target neuropilin-1, a receptor over-expressed in neovessels. Several modifications of the peptide were made and the analogues were tested through ELISA assays to evaluate their binding capability following the modifications. The third part of the thesis is related to the synthesis of porphyrin and peptide building blocks through click chemistry technique to form multiple conjugates with different numbers of porphyrins and peptides attached to the platforms

Despite the central role of IFNγ in vitiligo pathogenesis, systemic IFNγ neutralization is an impractical treatment option due to strong immunosuppression. However, most vitiligo patients present with less than 20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFNγ signaling during vitiligo, I hypothesized that tethering an IFNγ neutralizing antibody to keratinocytes would limit anti-IFNγ effects to the treated skin for the localized treatment. To that end, I developed a bispecific antibody (BsAb) capable of blocking IFNγ signaling while binding to desmoglein expressed by keratinocytes. I characterized the effect of the BsAb in vitro, ex vivo, and in a mouse model of vitiligo. SPECT/CT biodistribution and serum assays after local footpad injection revealed that the BsAb had improved skin retention, faster elimination from the blood, and less systemic IFNγ inhibition than the non-tethered version. Furthermore, the BsAb conferred localized protection almost exclusively to the treated footpad during vitiligo that was not possible by local injection of the non-tethered anti-IFNγ antibody. Thus, keratinocyte-tethering proved effective while significantly diminishing off-tissue effects of IFNγ blockade, offering a new treatment strategy for localized skin diseases, including vitiligo.

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No description available.

Anticancer active metal complexes such as cisplatin are routinely used for treating various cancers since 1978. However, the side effects of cisplatin overwhelm its therapeutic potential, especially in the latter stages of treatment. The nonspecific cytotoxicity of drugs could be avoided if targeted delivery to cancer cells is achieved using two different methodologies namely, enhanced permeability and retention in solid tumors (EPR) and receptor mediated endocytosis using a homing agent (RME). Ru(II)-arene complexes which are delivered specifically into cancer cells by the transferrin enzyme are less toxic compared to other metal complexes. The thesis describes the synthesis and use of Ru(II)-η6cymene complexes with different ancillary ligands which modulates the anticancer activity and the utility of two macromolecular vehicles in directed drug delivery. Ru(II)-η6cymene complexes with different heterocyclic ancillary ligands are synthesized and their anticancer activity tested against various cancer cell lines. Ruthenium complexes with mercaptobenzothiazoles are found to be quite active against the H460 cell lines that overexpress transferrin receptors and non-cytotoxic to the normal cell line, HEL299. Biophysical studies show that complexes (H1 and H8) can unwind the pBR322 DNA and inhibit the Topo IIα enzyme. A unique biphasic melting curve of CT DNA is observed in the presence of H1 which is attributed to formation of a dinuclear species (H20). Half-sandwich complexes of 6-thioguanine (6-TG) have also been prepared to improve the delivery and efficacy of 6-TG which is used in spite of a deleterious photoreaction. The Ru complexes cytotoxic to several leukemia cell lines. As they are photostable and anticancer active, they are better than 6-TG. Anticancer activity exhibiting piazselenols are used as ancillary ligands to make Ru(II)-arene complexes. Unfortunately, 1H NMR spectra suggests that piazselenol complexes dissociate in solution. However, the nitro substituted piazselenol and its Ru complex show the greatest cytotoxicity (<0.1 µM) against the A2780 cell line. The utility of PAMAM dendrimers and hyper branched polymers (hybramers) conjugated with a homing agent to target cancer cells by EPR and RME is probed. A cytotoxic copper complex (CuATSM) is covalently attached to the macromolecules through a disulfide linker, cleaved in the presence of GSH. Targeting efficacy of the folic acid-dendrimer conjugates is checked against two glioma cell lines. The folic acid-dendrimer conjugate is more active compared to dendrimer conjugate without folic acid against folate-receptor-overexpressing LN18 cell line. Biotin conjugated dendrimer shows better accumulation in HeLa cells, which require high amounts of biotin for growth. In vivo studies demonstrate that the conjugate can cross the blood-brain barrier. These studies suggest that PAMAM dendrimer can be used as a targeted delivery vehicle for cytotoxic metal complexes. Hyperbranched polymers decorated with propargyl groups and hydrophilic OH terminated TEG groups are attached to biotin and a cytotoxic Cu complex. (CuATSM-SS-CONH-N3) through ‘click’ reactions and tested against the HeLa cell line. On the basis of the studies conducted, it is concluded that targeted delivery of cytotoxic metal complexes are possible in the case of Ru(II) half-sandwich complexes and macromolecular vehicles like dendrimers are suitable for specifically delivering copper complexes into cancer cells.

O objetivo geral deste trabalho foi explorar a versatilidade de filmes multicamadas de polieletrólitos (PEM) e suas aplicações em sistemas de entrega de drogas e como filmes funcionais para aplicações biomédicas. Filmes PEM montados pela técnica de camada por camada (layer­-by­-layer, LbL), foram explorados em três aplicações principais. Na primeira, foi explorado o desenvolvimento de um protocolo de funcionalização em filmes de poli(alilamina)/poli (estireno sulfonato), PAH/SPS. Os parâmetros de construção do filme para biotinilação dos grupamentos amina do PAH foram otimizados e aplicados na captura e detecção do antígeno específico da próstata (PSA), na concentração de 100 a 0,1 ng/mL, usando pontos quânticos (Qdots). Em comparação com outros trabalhos, este sistema apresentou uma boa sensibilidade na detecção de PSA, dentro do limite de detecção clínica de 0,4 a 0,1 ng/mL. A segunda aplicação envolveu o desenvolvimento de filmes de sacrifício baseados nas interações naturais da mucina submandibular bovina e da lectina, jacalina (BSM/JAC). Filmes de BSM/JAC apresentaram estabilidade quando submetidos a uma ampla faixa de pH (pH 3-­-9) e em solução de alta força iônica (5 M NaCl). A dissolução dos filmes BSM/JAC pôde ser seletivamente desencadeada mediante à incubação em solução de melibiose, 37 °C, pH 7,4, sem apresentar citotoxicidade às células. Na última parte deste trabalho, a incorporação de lipossomos ecogênicos (ELIP) em mochilas celulares foi investigada. Mochilas celulares são \"patches\" de 7­-10 &#181;m de diâmetro que podem ser fabricados por meio de deposição alternada de polímeros utilizando-­-se a técnica de LbL, sobre uma matriz pré­-moldada obtida por fotolitografia, a fim de criar um sistema composto por três multicamadas estratificadas: uma região de liberação, para promover o destacamento do substrato, uma região de carga de droga, e uma região adesiva às células. O uso de ELIP permitiu incorporação de até 9x mais doxorrubicina (DOX) se comparado com o fármaco livre em solução absorvido pelos dos filmes. A liberação de DOX pelos filmes foi monitorado por 25 dias. Mochilas contendo ELIP-­DOX foram então aderidos a monócitos, e sua viabilidade monitorados por 72h. Mochilas vazias mostraram diminuir a proliferação de monócitos ao longo das 72 horas, enquanto mochilas carregadas com ELIP-­DOX mostraram uma diminuição dramática na população celular, apontando uma potencialização dos efeitos da droga pela sua proximidade com as células. / The overall goal of this thesis was to exploit the versatility of polyelectrolite multilayers (PEM) to be applied in drug delivery systems and biofunctionalizable films for biomedical applications. PEM films assembled by the layer-by­-layer technique were explored in three main applications. In the first part of this work, the development of a functionalization protocol of poly(allylamine)/poly(styrene sulfonate), PAH/SPS was explored. The optimal film parameters to the use of biotinylated multilayers were applied for the capture and detection of prostate specific antigen (PSA) protein in the range of 100 to 0.1 ng/mL, by using quantum dots. Compared to previous work, this system presented a good sensitivity for PSA detection that is within the clinical limit range of 0.4 to 0.1 ng/mL. The second application involved the creation of a novel sacrificial multilayer film. Films based in natural interactions of bovine submaxillary mucin and the lectin jacalin, BSM/JAC were assembled. BSM/JAC films showed stability when underwent a wide rage of pH (pH 3 to 9) and high ionic strength (5 M NaCl) solutions. BSM/JAC dissolution could be triggered released by incubation in melibiose at 37 °C in pH 7.4 buffer, without cytotoxicity. In the last part of this work the incorporation of echogenic liposomes (ELIP) into cell backpacks was investigated. Cell backpacks are 7-10 &#181;m diameter patches that can be fabricated through LbL polymer deposition onto a photopatterned array to create a stacked composite of three stratified multilayer systems: a releasable region for easy detachment from the substrate, a drug payload region, and a cell adhesive region. The use of ELIP allowed up to 9x more doxorubicin (DOX) loading when compared to free drug in solution adsorbed through the films. DOX release from films was monitored for over 25 days. ELIP­-DOX backpacks were then attached to mouse monocytes and their viability monitored by 72h. Empty backpacks showed to decrease monocytes proliferation over the course of 72h, while ELIP­-DOX backpacks showed a dramatic decrease in cell population, showing that DOX effects were enhancement in drug potency by its proximity.

Targeted delivery aids in minimizing most of the drug-originated systemic toxic effects as well as improving the pharmacokinetic properties of anticancer therapeutics. Tumor targeting using hyaluronic acid (HA) as the targeting ligand has attracted a great deal of interest among a host of strategies developed to target the overexpressed tumor specific receptors. HA is an endogenous molecule that possesses a lot of biological functions in the human body. The role of HA synthases, HA degrading enzymes and the interaction of HA with its primary receptor CD44 in tumor metastasis and angiogenesis is really complex and controversial to date. However, overexpression of CD44receptors on tumor surface has been well studied, which have been utilized to direct tumor targeted drugs. Most of the HA based targeting systems were HA drug conjugates and surface modified colloidal carriers which required covalent modification. The lack of accurate structural characterization of these systems resulted in modification of HA binding sites that could affect the efficient cellular uptake. LbL technique is a simple and facile method to incorporate several materials into polyelectrolyte assemblies for drug delivery applications. HA being a negatively charged polysaccharide can be easily incorporated into such systems without any covalent modification. Although HA based polyelectrolyte multilayer films and microcapsules have been reported in combination with polycations like PAH, PLL and chitosan, their application as targeted drug delivery systems have not yet been explored. Herein, two LbL architectures with HA as the terminal layer have been investigated as targeted drug carriers, which can recognize overexpressed CD44 receptors in metastatic breast cancer cells. In the first part of the thesis, a novel polyelectrolyte nanocapsule system composed of biopolymers HA and protamine sulphate (PR) as the wall components was prepared and characterized. These pH and enzyme responsive nanocapsules were then utilized for efficient loading and release of anticancer drug doxorubicin (dox). Higher drug release was observed in simulated intracellular conditions like acidic pH and presence of hyaluronidase enzyme as compared to physiological pH. In the second part of the thesis, dox incorporated bovine serum albumin (BSA) nanoparticles modified with HA-Poly(l-Lysine) multilayers were developed and characterized. The drug release pattern of the dox loaded BSA nanoparticles was found to depend on the presence of a protease enzyme trypsin than pH variations. Both of these drug delivery systems were then evaluated for their cell targeting efficiency and cytotoxicity in CD44+ positive metastatic breast cancer cell line MDA MB 231. The final layer HA facilitated targeted delivery of these drug carriers via CD44 receptor mediated endocytosis. The enhanced cellular uptake followed by sustained delivery of dox by virtue of slow intracellular enzymatic degradation of the drug carriers resulted in their improved cytotoxicity as compared to free dox. Further in vitro biodistribution and tumor suppression efficiency of both the systems were studied in breast cancer xenograft models using BALB/c nude mice. Enhance accumulation of dox in the tumor tissue and significant tumor reduction were observed when treated with encapsulated dox using the HA based nanocarriers as opposed to free dox.

O objetivo geral deste trabalho foi explorar a versatilidade de filmes multicamadas de polieletrólitos (PEM) e suas aplicações em sistemas de entrega de drogas e como filmes funcionais para aplicações biomédicas. Filmes PEM montados pela técnica de camada por camada (layer­-by­-layer, LbL), foram explorados em três aplicações principais. Na primeira, foi explorado o desenvolvimento de um protocolo de funcionalização em filmes de poli(alilamina)/poli (estireno sulfonato), PAH/SPS. Os parâmetros de construção do filme para biotinilação dos grupamentos amina do PAH foram otimizados e aplicados na captura e detecção do antígeno específico da próstata (PSA), na concentração de 100 a 0,1 ng/mL, usando pontos quânticos (Qdots). Em comparação com outros trabalhos, este sistema apresentou uma boa sensibilidade na detecção de PSA, dentro do limite de detecção clínica de 0,4 a 0,1 ng/mL. A segunda aplicação envolveu o desenvolvimento de filmes de sacrifício baseados nas interações naturais da mucina submandibular bovina e da lectina, jacalina (BSM/JAC). Filmes de BSM/JAC apresentaram estabilidade quando submetidos a uma ampla faixa de pH (pH 3-­-9) e em solução de alta força iônica (5 M NaCl). A dissolução dos filmes BSM/JAC pôde ser seletivamente desencadeada mediante à incubação em solução de melibiose, 37 °C, pH 7,4, sem apresentar citotoxicidade às células. Na última parte deste trabalho, a incorporação de lipossomos ecogênicos (ELIP) em mochilas celulares foi investigada. Mochilas celulares são \"patches\" de 7­-10 &#181;m de diâmetro que podem ser fabricados por meio de deposição alternada de polímeros utilizando-­-se a técnica de LbL, sobre uma matriz pré­-moldada obtida por fotolitografia, a fim de criar um sistema composto por três multicamadas estratificadas: uma região de liberação, para promover o destacamento do substrato, uma região de carga de droga, e uma região adesiva às células. O uso de ELIP permitiu incorporação de até 9x mais doxorrubicina (DOX) se comparado com o fármaco livre em solução absorvido pelos dos filmes. A liberação de DOX pelos filmes foi monitorado por 25 dias. Mochilas contendo ELIP-­DOX foram então aderidos a monócitos, e sua viabilidade monitorados por 72h. Mochilas vazias mostraram diminuir a proliferação de monócitos ao longo das 72 horas, enquanto mochilas carregadas com ELIP-­DOX mostraram uma diminuição dramática na população celular, apontando uma potencialização dos efeitos da droga pela sua proximidade com as células. / The overall goal of this thesis was to exploit the versatility of polyelectrolite multilayers (PEM) to be applied in drug delivery systems and biofunctionalizable films for biomedical applications. PEM films assembled by the layer-by­-layer technique were explored in three main applications. In the first part of this work, the development of a functionalization protocol of poly(allylamine)/poly(styrene sulfonate), PAH/SPS was explored. The optimal film parameters to the use of biotinylated multilayers were applied for the capture and detection of prostate specific antigen (PSA) protein in the range of 100 to 0.1 ng/mL, by using quantum dots. Compared to previous work, this system presented a good sensitivity for PSA detection that is within the clinical limit range of 0.4 to 0.1 ng/mL. The second application involved the creation of a novel sacrificial multilayer film. Films based in natural interactions of bovine submaxillary mucin and the lectin jacalin, BSM/JAC were assembled. BSM/JAC films showed stability when underwent a wide rage of pH (pH 3 to 9) and high ionic strength (5 M NaCl) solutions. BSM/JAC dissolution could be triggered released by incubation in melibiose at 37 °C in pH 7.4 buffer, without cytotoxicity. In the last part of this work the incorporation of echogenic liposomes (ELIP) into cell backpacks was investigated. Cell backpacks are 7-10 &#181;m diameter patches that can be fabricated through LbL polymer deposition onto a photopatterned array to create a stacked composite of three stratified multilayer systems: a releasable region for easy detachment from the substrate, a drug payload region, and a cell adhesive region. The use of ELIP allowed up to 9x more doxorubicin (DOX) loading when compared to free drug in solution adsorbed through the films. DOX release from films was monitored for over 25 days. ELIP­-DOX backpacks were then attached to mouse monocytes and their viability monitored by 72h. Empty backpacks showed to decrease monocytes proliferation over the course of 72h, while ELIP­-DOX backpacks showed a dramatic decrease in cell population, showing that DOX effects were enhancement in drug potency by its proximity.

Chemotherapy is still one of the most widely used anticancer therapies. It is mostly about inhibiting the proliferation of rapidly dividing cells, so it is not selective for tumor cells. As a result, many undesirable side effects are associated with chemotherapy. The disadvantageous properties of chemotherapeutics can be largely eliminated by using conjugates of polymers with low molecular weight drugs. An example of such a conjugate is a doxorubicin-linked HPMA polymer. In addition to the properties obtained by polymer binding, such as achieving solubility in aqueous solutions, reducing systemic toxicity, increasing the maximum tolerated dose, or passive targeting by the EPR effect, the fact that doxorubicin induces immunogenic cell death is used in therapy with this drug. It has already been shown that after complete cure of the experimental mice with polymeric conjugates of HPMA with doxorubicin, some mice develop long-term resistance to re-inoculation with a lethal dose of tumor cells. Resistance is specific to the particular line of tumor cells from which the mouse was cured, and CD8+ cytotoxic T cells and IFNγ play an important role. In this work, we monitored changes in the proportion of immune populations and their activation markers after treatment with HPMA-based polymers with doxorubicin...

A prevalent morphology in the microscopic world of artificial microswimmers, bacteria and viruses is that of a helix. The intriguingly different physics at play at the small scale level make it necessary for bacteria to employ swimming strategies different from our everyday experience, such as the rotation of a helical filament. Bio-inspired microswimmers that mimic bacterial locomotion achieve propulsion at the microscale level using magnetically actuated, rotating helical filaments. A promising application of these artificial microswimmers is in non-invasive medicine, for drug delivery to tumours or microsurgery. Two crucial features need to be addressed in the design of microswimmers. First, the ability to selectively control large ensembles and second, the adaptivity to move through complex conduit geometries, such as the constrictions and curves of the tortuous tumour microvasculature. In this dissertation, a mechanics-based selective control mechanism for magnetic microswimmers is proposed, and a model and simulation of an elastic helix passing through a constricted microchannel are developed. Thereafter, a theoretical framework is developed for the propulsion by stiff elastic filaments in viscous fluids. In order to address this fluid-structure problem, a pertubative, asymptotic, elastohydrodynamic approach is used to characterise the deformation that arises from and in turn affects the motion. This framework is applied to the helical filaments of bacteria and magnetically actuated microswimmers. The dissertation then turns to the sub-bacterial scale of bacteriophage viruses, 'phages' for short, that infect bacteria by ejecting their genetic material and replicating inside their host. The valuable insight that phages can offer in our fight against pathogenic bacteria and the possibility of phage therapy as an alternative to antibiotics, are of paramount importance to tackle antibiotics resistance. In contrast to typical phages, flagellotropic phages first attach to bacterial flagella, and have the striking ability to reach the cell body for infection, despite their lack of independent motion. The last part of the dissertation develops the first theoretical model for the nut-and-bolt mechanism (proposed by Berg and Anderson in 1973). A nut being rotated will move along a bolt. Similarly, a phage wraps itself around a flagellum possessing helical grooves, and exploits the rotation of the flagellum in order to passively travel along and towards the cell body, according to this mechanism. The predictions from the model agree with experimental observations with respect to directionality, speed and the requirements for succesful translocation.