Biossensores que empregam dispositivos de efeitos de campo podem ser obtidos em diversas arquiteturas, incluindo dispositivos Eletrólito-Isolante-Semicondutor (EIS), que são capacitores em que o eletrodo metálico é substituído por um filme e uma solução. Medindo-se a capacitância em função do potencial aplicado, é possível detectar variações de pH oriundas de reações ou interações entre o filme e o analito. Nesta dissertação, sensores foram produzidos com a adsorção de filmes automontados de dendrímero (PAMAM) e nanotubos de carbono (SWNT) num chip. Medidas de espectroscopia de impedância foram realizadas para investigar o crescimento de cada bicamada do filme automontado, e os dados foram analisados com circuitos equivalentes que continham uma capacitância de dupla camada, um elemento de fase constante e uma capacitância para a região de depleção. Para o chip, os melhores ajustes foram obtidos na frequência de 2 kHz, em que a concentração de dopantes foi 6,6x1020 m-3 para o chip com isolante de SiO2 e de 1,1x1021 m-3 para o chip com isolante de SiO2/Ta2O5. O potencial de banda plana foi -0,2 V e -0,06V, respectivamente. Para os chips recobertos com os filmes de PAMAM/SWNT, observamos que a região de depleção é causada pelas cargas positivas do PAMAM. Com relação às implicações para biossensores, verificamos que o desempenho ótimo deve ser obtido com 3 bicamadas de PAMAM/SWNT. Isso pode explicar a observação empírica na literatura de que existe uma espessura ideal dos filmes para um desempenho otimizado. / Biosensors based on field effect devices can be produced with several architectures, including Electrolyte-Insulator-Semiconductor (EIS) devices, which are capacitors where conventional metal electrodes are replaced by a sensing layer and an electrolyte solution. By measuring the capacitance as a function of the bias voltage, it is possible to detect pH changes that may originate from reactions or interactions between the film in the sensing unit and the analyte. In this study sensors were obtained by adsorbing layer-by-layer (LbL) films made with dendrimers (PAMAM) and carbon nanotubes (SWNT) on a semiconductor chip. Impedance spectroscopy measurements were performed to monitor the growth of each bilayer in the LbL film, whose data were analyzed with equivalent circuits containing a double-layer capacitance, a constant phase element and a capacitance for the depletion region. The results for the semiconductor chip could be best fitted for a frequency of 2 kHz, where the doping concentration was 6.6 x1020 m-3 for the insulating SiO2 layer and 1.1 x1021 m-3 for the SiO2/Ta2O5 layer. The flat band voltage was -0.2 V and -0.06 V, respectively. In the analysis of the chip coated with different numbers of PAMAM/SWNT bilayers, we found that the depletion region appears as a contribution from the positive charges in the PAMAM layer. With regard to implications for biosensors, we found that optimized performance should be reached with three PAMAM/SWNT bilayers, which may explain the empirical finding in the literature that an ideal thickness exists for enhanced performance.

Dendrimers are a class of polymers with a highly branched, three-dimensional architecture composed of an initiator core, several interior layers of repeating units and multiple surface groups. They have been recognized as the most versatile compositionally and structurally controlled nanoscale building blocks throughout the fields of engineering, materials science, chemistry, and biology, and they have been widely investigated for drug and gene delivery. Polyamidoamine (PAMAM) dendrimers have inherent properties for gene delivery because of their high buffering capacity, polycationic surface and numerous surface groups for biofunctionlization. This dissertation is organized into four independent sections. The first section investigates a series of polyamidoamine-polyethylene glycol-poly (D,L-lactide) (G3.0- PEG1500-PDLLA, G3.0-PEG6000-PDLLA, and G3.0-PEG12000-PDLLA) for gene delivery. Western Blot, fluorescence microscopy and flow cytometry were used as analysis methods. According to gene transfection studies, G3.0-PEG1500-PDLLA has been shown to be capable of inducing higher gene expression than the parent dendrimer compared to unmodified dendrimer, G3.0-PEG6000-PDLLA and G3.0-PEG12000- PDLLA. The second section aims to evaluate an epidermal growth factor (EGF)-containing PAMAM G4.0 dendrimer vector labeled with quantum dots for targeted imaging and nucleic acid delivery. Targeting efficiency, cell viability, proliferation, and intracellular signal transduction were evaluated. We found that EGF-conjugated dendrimers did not stimulate growth of epidermal growth factor receptor (EGFR)-expressing cells at the selected concentration. Consistent with this, minimal stimulation of post-receptor signaling pathways was observed. These nanoparticles can localize within cells that express the EGFR in a receptor-dependent manner, whereas uptake into cells lacking the receptor was low. Vimentin short hairpin RNA (shVIM) and yellow fluorescent protein (YFP) small interfering RNA (siRNA) were used to test the delivery and transfection efficiency of the constructed targeted vector. Significant knockdown of expression was observed, indicating that this vector is useful for introduction of nucleic acids or drugs into cells by a receptor-targeted mechanism. The third section introduces PEGylated polyamidoamine (PAMAM) dendrimer G4.0 conjugates with a novel bis-aryl hydrazone (BAH) linkage for gene delivery. It was found that the incorporation of BAH linkages into the vector significantly enhanced the buffering capacity of the vector with a high degree of PEGylation. According to gene transfection studies, this new vector has been shown to be capable of both transfecting more cells and inducing higher gene expression than the parent dendrimer. This work demonstrates that the use of the BAH linkage in coupling of PEG to the dendrimer helps maintain or increase the buffering capacity of the functionalized dendrimer and results in enhanced transfection. In the fourth section, we explored PAMAM dendrimer G4.5 as the underlying carrier to construct central nervous system (CNS) therapeutic nanoparticles and tested the buccal mucosa as an alternative absorption site for administration of the dendritic nanoparticles. Opioid peptide DPDPE was chosen as a model CNS drug. It was coupled to PAMAM dendrimer G4.5 with PEG or with PEG and transferrin receptor monoclonal antibody OX26. The therapeutic dendritic nanoparticles labeled with 5-(aminoacetamido) fluorescein (AAF) or fluorescein isothiocyanate (FITC) were studied for transbuccal transport using a vertical Franz diffusion cell system mounted with porcine buccal mucosa. Coadministration of bile salt sodium glycodeoxycholate (NaGDC) or application of mucoadhesive gelatin/PEG semi-interpenetrating network (sIPN) enhanced the permeability of dendritic nanoparticles by multiple folds. These results indicate that transbuccal delivery is a possible route for administration of CNS therapeutic nanoparticles. In summary, enhanced nucleic acids delivery by biofunctionalized PAMAM dendrimers was demonstrated. Transbuccal delivery of CNS therapeutic dendritic nanoparticles was demonstrated. These vectors will be useful in gene and drug delivery and could be extended to covalently conjugate other functional moieties for gene and drug delivery.

Anticancer therapeutics are often limited to suboptimal doses due to their lack of selectivity for tumor cells and resultant damage to healthy tissue. These limitations motivated researchers to develop tumor-specific delivery systems for improved therapeutic efficacy and reduced unintended cytotoxicity. Polyamidoamine dendrimers offer an ideal platform for designing targeted therapeutics with tunable characteristics that optimize pharmacokinetic behavior and targeting specificity. Ligand conjugation to dendrimer provides the biochemical interaction necessary to activate tumor-specific receptors for receptor-mediated endocytosis and effective internalization of polyplexes. Tumor-specific receptors overexpressed in carcinomas, like folate receptor-alpha (FOLRα), are targeted by ligand-conjugated dendrimer to allow enhanced internalization of dendrimer and its therapeutic cargo. We examined the cellular trafficking dynamics and potential of folate-conjugated dendrimer for nucleic acid delivery in vitro. Results show folate-conjugation to G4 PAMAM dendrimer (G4FA) confers enhanced uptake in FOLRα-positive tumor cells. Cells internalize G4FA in a receptor-dependent manner with specificity for FOLRα-positive tumor cells.

L'efficacité de la thérapie photodynamique (PDT) est confrontée à plusieurs verrous : les photosensibilisateurs (PSs) utilisés en clinique ne sont pas adaptés à la fenêtre thérapeutique, ils subissent un photoblanchiment lors du traitement, et leur nature organique pose des problèmes de solubilité en milieu biologique. Ils présentent également une faible sélectivité envers les tissus tumoraux à traiter. Dans le cadre de cette thèse, trois approches visant une amélioration de la PDT appliquée à la cancérologie ont été développées : la vectorisation, l'adressage, et l'optimisation de nouveaux PSs. La synthèse de structure PS/vecteur a permis d'élaborer des structures hydrophiles capables de vectoriser des PSs hydrophobes. Des porphyrines ont ainsi été greffées sur des dendrimères polyamidoamine (PAMAM) dissymétriques. La conservation des propriétés photophysiques des PSs après leur couplage au dendrimère a été mise en évidence. Des quantum dots (QDs), grâce à la modularité de leurs propriétés photophysiques et leur capacité à résister au photoblanchiment, ont été synthétisés et utilisés comme nouvelle classe d'agents photosensibilisants. Ces QDs ont été préparés afin d'être hydrophiles et utilisables dans la fenêtre thérapeutique de la PDT. Une étude in vitro des QDs couplés à l'acide folique a mis en évidence leur activité photodynamique. Des études réalisées par une approche de plans d'expérience a permis de hiérarchiser les facteurs expérimentaux en fonction de leurs impacts sur l'activité photodynamique. Nous avons en particulier montré une amélioration de la sélectivité des conjugués envers les cellules surexprimant le récepteur à l'acide folique.

The research in this dissertation examines the chemistry and applications of dendrimers in homogeneous catalysis. We examined interactions between dendrimers and charged probe molecules, prepared dendrimer-encapsulated metal nanoparticles in organic solvents, studied size-selectivity of dendrimer-encapsulted catalysts, and designed molecular rulers as in-situ probes to measure the location of dendrimer-encapsulted metal nanoparticles. The intrinsic proton binding constant and a constant that characterizes the strength of electrostatic interactions among occupied binding sites in poly(amidoamine) (PAMAM) dendrimers have been obtained by studying the effect of solution pH on the protonation of the dendrimers. The significant finding is that these two factors are greatly modulated by the unique and hydrophobic microenvironment in the dendrimer interior. Hydrophilic poly(propylene imine) (PPI) dendrimers were modified with various hydrophobic alkyl chains through an amide linkage and were then used as templates for preparing intradendrimer copper nanoclusters. The main driving force for encapsulating metal-ions was found to be the differences in metal-ion solubility between the solvent and the interior of the dendrimer. Nanometer-sized metal particles are synthesized and encapsulated into the interior of dendrimers by first mixing together the dendrimer and metal ion solution and then reducing the composite chemically, and the resulting dendrimer-encapsulated metal nanoparticles can then be used as catalysts. By controlling the packing density on the dendrimer periphery using either different dendrimer generations or dendrimer surface functionalities, it is possible to control access of substrates to the encapsulated catalytic nanoparticle. Molecular rulers consisting of a large molecular "stopper", a reactive probe and a linker were designed as in-situ probes for determining the average distance between the surface of dendrimer-encapsulated palladium nanoparticles and the periphery of their fourth-generation, hydroxyl-terminated PAMAM dendrimer hosts. By doing so, we avoid having to make assumptions about the nanoparticle size and shape. The results suggest that the surface of the encapsulated nanoparticle is situated 0.7 ± 0.2 nm from the surface of the dendrimer.

Le travail réalisé au cours de cette thèse a porté sur le développement de biocapteurs électrochimiques d’affinité, sensibles et sélectifs, pour la détection de l’ochratoxine A (OTA) et l’aflatoxine M1 (AFM1). Les biocapteurs développés reposent sur l’association de différents nanomatériaux pour une meilleure performance analytique. Pour construire notre transducteur, nous avons associé le polypyrrole à des dendrimères poly(amido-amine) PAMAM, ce qui a permis d’avoir de très bon rendements grâce au propriétés électriques du polypyrrole et à l’augmentation de la surface active due à la structure tridimensionnelle des dendrimères. L’utilisation d’aptamères spécifiques pour la détection des différentes mycotoxines a permis leur détection et quantification à des concentrations de l’ordre des nM, ainsi que l’élargissement des gammes dynamiques. Nous avons pu démontrer grâce à l’utilisation de dendrimères de différentes tailles que la sensibilité des biocapteurs ne provient pas uniquement de l’affinité qui existe entre les biorécepteurs et leurs molécules cibles, mais aussi des propriétés physico-chimiques du biocapteur. / This aim of this work is to develop ultrasensitive electrochemical biosensors with high affinity toward ochratoxine (OTA) and aflatoxine M1 (AFM1). In order to obtain the best analytical performances, we associated nano-materials in the transducer construction: conducting polypyrrole polymer and poly(amido-amine) dendrimères. Thanks to this association, we benefited from the conducting material’s electrical properties, and the large active detection surface dendrimers. For the bimolecular sensing part, we used specific DNA aptamers which allowed us to quantify mycotoxines at nM concentrations. In addition, the different aptamer based biosensors present a very large dynamic ranges. We also demonstrated through the use of different sizes of dendrimers, that the sensitivity depend not only in the affinity between bioreceptors and their target molecules, but also in the physico-chemical properties of the biosensor.

Thin film Nanocomposite (TFN) membranes are a relatively new class of high-performance semipermeable membranes for Reverse Osmosis (RO) applications. Large scale applications of TFN membranes have not been achieved yet due to the high production cost of the nanoparticles, agglomeration of the nanoparticles in the thin polyamide matrix of the membrane, and leaching out of typically toxic inorganic nanoparticles into the downstream. In this work, these challenges are addressed by incorporation of two different nanofillers: Cellulose NanoCrystals (CNC), and surface functionalized Halloysite NanoTubes (HNT). Amine groups, carboxylic acid groups, and the first generation of poly(amidoamine) (PAMAM) dendrimers were used for functionalization of the HNT. CNC and HNT are environmentally friendly, low/non-toxic, abundant, and inexpensive nanoparticles with a unique size, and chemical properties. TFN membranes were synthesized via in situ interfacial polymerization of m-phenylenediamine (MPD) with trimesoyl chloride (TMC) and the nanoparticles. The control Thin Film Composite (TFC) membranes, and CNC and HNT based TFN membranes were characterized by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), X-Ray Diffraction (XRD), X-ray Photoelectron Spectroscopy (XPS), Fourier Transform Infrared spectroscopy (FTIR) and contact angle measurements. The antifouling capacity of CNC based membranes was investigated with a solution of Bovine Serum Albumin (BSA) as the fouling agent. Also, the leachability of the HNT from the membranes was examined by shaking the membranes in a batch incubator for 48 h, and then tracing the leached out HNT using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Separation characteristics of the membranes were studied by desalination of synthetic brackish water with a cross flow RO filtration system. It was revealed that incorporation of functionalized HNT enhanced the permeate flux without sacrificing the salt rejection (99.1 % ± 0.1 %). Also, incorporation of 0.1% (w/v) CNC doubled the permeate flux (from 30 to 63 L/m2.h at 20 bar) without compromising the salt rejection (97.8%). At the same time, leaching out of HNT from the TFN membranes was decreased as a result of the HNT functionalization and formation of covalent bonds with the TMC. Also, antifouling properties of the CNC-TFN membranes were 11% improved in comparison with control TFC membrane.

Content: Environmental pollution caused by leather making is the primary concern in the development of leather industry. The use of safe, effective and multi-functional green chemical products has the advantages of reducing leather operations, increasing chemicals utilization, decreasing the environmental burden, improving leather quality. In this study, dendritic-linear polymers of PAMAM-Si 1G and PAMAM-Si 2G were applied to fatliquoring process, which were prepared by branching polysiloxane on the dendritic polyamide-amine (PAMAM). Then the emulsion properties, fatliquoring properties and fatliquoring mechanism were studied by EDS, SEM, XRD, TG and washing experiments. The conclusion was drawn that PAMAM-Si are weak alkali products with high emulsion stability. The particle size of PAMAM-Si 1G was 35.8 nm, and that of PAMAM-Si 2G was 26.7 nm. They can improve the softness, shrinkage temperature and physical and mechanical properties of leather. The softness of leather with PAMAM-Si 1G and PAMAM-Si 2G increased by 115.6% and 104.7% respectively. The shrinkage temperature of leather with PAMAM-Si 2G increased by 2.9℃. The Breaking elongation of leather with PAMAM-Si 1G and PAMAM-Si 2G increased by 38.6% and 32.4% respectively. At the same time, PAMAM-Si not only increased the distance and disorder of fiber but combined with collagen fiber through hydrogen bond, a certain amount of physical adsorption and covalent bond. Take-Away: 1. The dendritic-linear polymers of PAMAM-Si 1G and PAMAM-Si 2G were prepared by branching polysiloxane on the dendritic polyamide-amine (PAMAM). 2. PAMAM-Si can improve the softness, shrinkage temperature and physical and mechanical properties of leather. 3. PAMAM-Si not only increased the distance and disorder of fiber but combined with collagen fiber through hydrogen bond, a certain amount of physical adsorption and covalent bond.

Philosophiae Doctor - PhD / Introduction: Tuberculosis (TB) is a serious bacterial infections caused by the Mycobacterium Tuberculosis (MTB) organism affecting mainly the lungs. Occasionally, MTB bacilli may be transported out of the pulmonary region and infect peripheral organs causing extra-pulmonary tuberculosis. Many therapeutic agents were developed over the years to combat TB, however the rapid emergence of resistant strains hampered their use. Furthermore, most of the current anti-TB drugs experience many challenges, which can be summarized in treatment regimen factors, drug-drug interactions, and physicochemical characteristics factors (such as hydrophobicity and low permeability into alveolar macrophages). These challenges have a significant role in treatment failure and the emergence of resistant TB. Due to the lack of newly discovered anti-TB drugs, and the absence of effective vaccines, many scientists have suggested the use of novel modalities for the current anti-TB drugs to enhance their efficacy and overcome some of the drawbacks. One of these modalities is nanotechnology-based drug delivery systems. Most of the anti-TB drugs experience low drug distribution to the lung and particularly alveolar macrophages within which the MTB resides, leading to treatment failure. Employing nanoparticles as drug delivery systems can have a significant impact on improving the pharmacokinetic profile of anti-TB drugs, the feasibility of different routes of administration, enhancing drug permeability, controlled/sustained drug release, and targeting specific disease sites. Collectively, these impacts will aid in enhancing drug concentration at the site of infection and reduce dosing and regimen duration. Dendrimers, such as polyamidoamine (PAMAM) dendrimers, are synthetic polymeric nanoparticles that have unique features that afford a dendrimer-conjugate complex the possibility to overcome the most common hurdles associated with drug delivery and treatment of diseases. Obstacles associated with solubility, permeability, inadequate biodistribution associated side effects may be enhanced. Manipulating the outermost surface functional groups with various ligands and polymers, will enhance the dendrimer properties and targeting potential. Aim: This study aims to develop a novel pulmonary delivery system for the anti-TB drug rifampicin using surface-modified G4 PAMAM dendrimer nanoparticles (polyethylene glycol (PEG) or mannose moieties), to improve drug solubility, prolong-release, enhance permeability into the macrophages, and decrease the toxicity of the drug-dendrimer conjugates. Methods: PAMAM dendrimers having increasing concentrations of poly(ethylene glycol) (PEG) 2 kDa or mannose residues were synthesized. The 4-nitrophenyl chloroformate was used as an activator in the case of PEG functionalization, while for the mannose conjugation the 4-isothiocyanatophenyl alpha-D-mannopyranoside (4-ICPMP) directly interacted with the primary amines of the dendrimer. The conjugated PEG polymers and mannose moieties on the dendrimer periphery were confirmed using FTIR and 1H NMR analytical techniques. Thereafter, rifampicin was loaded into the native and surface-modified dendrimers via a simple dissolution solvent evaporation method. Rifampicin-loaded dendrimers were then characterized using several analytical techniques namely; FTIR, DSC, NMR, SEM, and DLS. The polymer encapsulation efficiency (EE%) and percentage of drug loading (DL%) were determined directly using a validated HPLC method. In vitro drug release was studied at pH 7.4 and pH 4.5. The MTT technique was used to assess the cytotoxicity of the dendrimer formulations against raw 264.7 cell lines. Finally, the uptake of dendrimer nanoparticles by raw macrophages was studied using a flow cytometer and fluorescence microscopy techniques. Results: The percentage coverage of 4.0 G PAMAM dendrimer peripheral with PEG was achieved in a range of 38% - 100%, while for mannose moieties was from 44% - 100%. The EE% of unmodified dendrimer was 7.5% (w/w). The EE% of PEGylated dendrimers ranged from 65.0% - 78.75% (w/w), whereas for mannosylated dendrimers was from 43.43% - 57.91% (w/w). The size of the unloaded dendrimer nanoparticles was less than 25 nm, a gradual increase in the size after drug conjugation followed. The zeta potential of dendrimers was positive with values greater than 12 mV, the nanoparticle's zeta potential decreased upon increasing the density of PEG/mannose and after drug loading. FTIR and NMR data showed that rifampicin molecules were conjugated to the dendrimer at three sites; at the surface amines via electrostatic linkages, within the PEG/mannose, and into the dendrimer interior. SEM images of dendrimer nanoparticles confirmed the spherical shape of particles, and DSC data verified drug entrapment. Drug release was found to be affected by the pH of the medium and the extent of dendrimer functionalization. At the physiologic pH, surface-modified dendrimers showed a slower release rate compared to the unmodified dendrimer and free drug. Among surface-modified dendrimers, the release rate was inversely associated with the density of PEG/mannose molecules. At pH 4.5, a relatively higher drug release from all formulations was observed which suggests a burst release inside the alveolar macrophages. Toxicity studies showed that the unmodified dendrimer experienced time-dependent and concentration-dependent cytotoxicity against raw 264.7 cells. The toxicity gradually decreased upon increasing the density of PEG/mannose, and negligible toxicity was detected for formulations with 100% functionalization. Dendrimer nanoparticles were successfully internalized into raw cells after 24 hrs of incubation. The order of nanoparticles permeability was PEG 100% < PEG 85% < PEG 70% < PEG 49% < PEG 38% < unmodified dendrimer < mannose 44% < mannose 69% < mannose 93% < mannose 100%. The significant increase in the uptake of mannosylated dendrimers was due to the interaction with lectin receptors at the surface of raw macrophages, whereas the lower internalization of PEGylated dendrimers was due to the shielding of the surface positive charges. Conclusion: The in-vitro and ex-vivo data studies suggested that the developed novel surface-modified G4 PAMAM dendrimers are suitable drug carriers in terms of biocompatibility, release behaviour, and site-specific delivery of the anti-TB drug rifampicin.

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