Human α-N-acetylglucosaminidase (Naglu) is a lysosomal enzyme implicated in the rare metabolic storage disorder Mucopolysaccharidosis III type B (MPS IIIB). A deficiency in Naglu results in a buildup of heparan sulfate in lysosomes, which is most detrimental in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is currently ineffective in treating the neurological symptoms of MPS IIIB due to the inability of Naglu to cross the blood-brain barrier. This laboratory uses a Spodoptera frugiperda insect cell system to express recombinant Naglu conjugated to a synthetic protein transduction domain with the intent to allow Naglu to cross the blood-brain barrier and treat the neurological symptoms. In the present study, we aimed to purify a recombinant Naglu-PTD4 fusion protein in order to assess its capacity to cross cellular membranes. A three-step method involving multi-modal, hydrophobic interaction, and gel filtration chromatography was optimized to achieve pure Naglu-PTD4, in good yield. Cellular uptake by human MPSIIIB fibroblasts of Naglu-PTD4 was not detectable. It is hypothesized that additional amino acids, including a hexahistidine domain, following the PTD4 domain limited the fusion protein’s membrane transduction capacity. Future studies will focus on removing the additional amino acids and adjusting the purification method as necessary. The ultimate goal of this research is to develop a large-scale recombinant Naglu production protocol for enzyme replacement therapy of MPS IIIB. / Graduate

Liposomal ciprofloxacin (Lipo-CPFX) is being developed for inhalation, with a goal of sustaining the therapeutic activity, compared to unformulated ciprofloxacin (CPFX). However, the kinetics and mechanism of its sustained local lung retention and pharmacological activity are yet to be fully characterized. This project hypothesized that Lipo-CPFX enables slower and sustained lung epithelial transport and uptake, compared to CPFX, thereby producing prolonged local pharmacological actions. The human bronchial epithelial Calu-3 cells were used as monolayers to characterize the kinetics and mechanism of transport and/or uptake, and to assess the effects of such slow kinetics for Lipo-CPFX on its inhibition against lipopolysaccharide (LPS)-induced proinflammatory IL-8 release. The transport fluxes for Lipo-CPFX across the highly restricted Calu-3 cell monolayers was transepithelial electrical resistance-independent, which suggested predominant transcellular transport. Compared to CPFX, Lipo-CPFX showed 6-18 times slower transport, while the flux was increased with increasing concentration proportionally without saturation. Its unaltered transport by cellular energy depletion, transport inhibition by a reduced temperature (4 oC) and endocytosis/lipid fusion inhibitors, filipin and LysoPC, and increased transport by excess empty liposomes collectively suggested cell energy-independent, lipid bilayer fusion mechanisms for the Lipo-CPFX transport across the Calu-3 cells. Likewise, Lipo-CPFX showed 2-4 fold lower cellular uptake than CPFX, proportional to concentration. Lipo-CPFX exhibited significant inhibitory activities at ≥ 0.01 mg/mL on LPS-induced IL-8 release from the Calu-3 cells, which was equipotent to CPFX. Upon 24 h pre-incubation, Lipo-CPFX caused 36.9 and 47.5 % inhibition at 0.01 and 0.05 mg/mL, respectively, while CPFX failed to do so. However, the effect was negated upon repeated wash of the mucosal cell surface, speculating the importance of cell membrane-associated drug/formulation on the inhibitory activities for Lipo-CPFX. Upon 24 h transport, Lipo-CPFX retained 79.0 % of the 4 µg dose on the mucosal cell surface, which was 1.9-times greater than 40.7 % for CPFX. As a result, when LPS was added at 24 h of the transport, Lipo-CPFX was still capable of causing 60.1 % inhibition, as its sustained local anti-inflammatory activity; CPFX however also exhibited equipotent inhibition, by virtue of comparable cellular drug uptake/transport.

Les nanotechnologies sont en pleine expansion et leurs propriétés remarquables ouvrent la voie à des applications très variées intéressant la science des matériaux comme la nanomédecine. Cependant cet essor fulgurant doit s’accompagner d’interrogations justifiées sur les risques sanitaires pour l’homme et l’environnement compte tenu de leur potentielle toxicité biologique. Dans ce contexte le travail de cette thèse porte sur l’amélioration de la compréhension des mécanismes d’internalisation de particules au niveau cellulaire. Pour cela plusieurs types de micro- et nanoparticules fluorescentes de physico-chimie contrôlée (taille et groupements de surface) ont été synthétisés. Des contacts ont ensuite été établis avec une lignée cellulaire in vitro de macrophages (RAW 264.7) qui représente une cible préférentielle de l’organisme au niveau des moyens de défense. Différentes méthodologies de quantification en cytométrie en flux et fluorimétrie ont été développées dans le but de distinguer précisément les nanoparticules internalisées de celles adhérées au niveau des membranes. Des techniques complémentaires de microscopie confocale et MEB/MET ont été mises au point afin de mieux visualiser leur localisation intracellulaire. Enfin, pour nos diverses conditions expérimentales, une étude de l’activité biologique des particules a été évaluée sur la base des paramètres de réaction inflammatoire, d’altération membranaire et de stress oxydant. Ainsi, la première partie de ces travaux a concerné la mise au point d’une méthodologie de quantification de l’internalisation de particules fluorescentes microscopiques. Ensuite nous avons développé un modèle de nanoparticules doublement fluorescentes sensibles aux variations de pH permettant une quantification plus ciblée du processus de phagocytose. Enfin la dernière partie s’est attachée à étudier spécifiquement l’impact de la taille des nanoparticules sur leur internalisation. / Nanotechnologies are in full extension and the remarkable properties showed by nanomaterials pave the way for a variety of applications such as materials science or nanomedicine. However, this rapid expansion must be accompanied by justified interrogations about human’s health risks or impact on the environment because of their potential biological toxicity. In this context, this thesis aimed at improving the understanding of the cellular internalization mechanisms of particles. Different types of fluorescent micro- and nanoparticles with well-controlled physico-chemistry (size and surface groups) were synthesized. The contacts were established with an in vitro macrophage cell line (RAW 264.7), which represents the first line target cells in the human defense mechanisms. Different methodologies to accurately quantify and distinguish internalized nanoparticles from those just adhering to cell membranes were developed using flow cytometry and fluorimetry. Complementary confocal microscopy and SEM/TEM techniques were carried out to better visualize nanoparticles intracellular localization. Finally, for each experimental condition tested, the biological activity of the particles was evaluated in terms of inflammatory response, membrane alteration and oxidative stress. Thus, the first part of this work allowed the development of a methodology to quantify fluorescent microscopic particles internalization. Then a model of double fluorescent nanoparticles sensitive to pH variations was developed in order to quantify more precisely the phagocytic process. Finally the last part aimed at evaluating the impact of the size of the nanoparticles on their internalization.

Orientador: Prof. Dr. Fernando Carlos Giacomelli / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2015. / O presente estudo se insere na emergente area da nanomedicina, tendo como base a ciencia de coloides e interfaces, e teve como objetivo inicial a sintese de nanoparticulas polimericas usando diferentes copolimeros para se obter sistemas nanoestruturados com diferentes caracteristicas no que se refere a dimensao, carga superficial e estrutura interna. Os dados experimentais de espalhamento de luz e imagem mostraram que foi possivel obter sistemas polimericos nanoestruturados de diferentes tamanhos e carga superficial utilizando o protocolo de nanoprecipitacao. Haja vista a diferenca estrutural dos polimeros utilizados, as nanoparticulas produzidas sao estruturalmente diferentes do ponto de vista de tamanho de nucleo hidrofobico e coroa hidrofilica. Tambem foi possivel elucidar a influencia destes parametros estruturais na captura e internalizacao celular das entidades. Os resultados sugerem que a espessura da coroa hidrofilica de polioxietileno (PEO), que tem o papel fundamental de estabilizar as nanoestruturas produzidas, e decisiva no processo. Na medida em que foram utilizados copolimeros em bloco de longa cadeia hidrofilica, percebeu-se uma reducao semi-quantitativa e quantitativa na internalizacao celular das entidades. A carga superficial tambem possui importante papel no processo que, porem, e ofuscado pelo fator dimensao da coroa, onde para nanoparticulas de elevado potencial-¿ê (~ - 40 mV), que e o caso de NPs de PLGA, a internalizacao celular nao apresentou diferenca estatisticamente significativa em comparacao com outros sistemas. Por outro lado, NPs apresentando cadeias estabilizantes de PEO mais curtas (Mw ~ 2000 g.mol-1) foram mais eficientemente internalizadas, apesar de seu potencial-¿ê negativo (~ -20 mV). Para NPs com o mesmo tamanho de cadeia estabilizante de PEO, os dados experimentais sugerem que NPs formadas por um bloco hidrofobico de policaprolactona (PCL) sao internalizadas de maneira mais eficiente em comparacao com NPs formadas por um bloco hidrofobico de poli acido latico (PLA). Este ultimo efeito foi atribuido a maior hidrofobicidade do bloco de PCL, o que vai de encontro com o modelo teorico da molhabilidade, apesar dos resultados de SPR mostrar adsorcao a membrana mimetica somente das NPs a base de PLA. / Herein, the cellular uptake of naked PLGA and PLA- and PCL-based block copolymer nanoparticles (NPs) have been evaluated. The nanoparticles were produced containing the same amount of loaded fluorescent cumarina-6 (0.5 % wprobe/wpolymer) by using the so-called nanoprecipitation protocol. This procedure yielded nano-sized objects with distinct structural features dependent on the length of the hydrophobic and hydrophilic blocks and volume ratio. They were further detailed characterized by scattering techniques and atomic force microscopy. The cellular uptake of selected nanoparticles has been evaluated by laser confocal scanning microscopy and flow cytometry analysis. The cellular uptake events were examined in relation to size, surface charge, hydrophobic core nature and hydrophilic chain length of the produced NPs. The experimental results indicated that cellular uptake was considerably enhanced as the length of the hydrophilic PEO-stabilizing shell reduces. Additionally, it has been also evidenced that a high negative surface charge restricts cellular uptake and that, on the other hand, nanoparticles comprising a hydrophobic core of higher degree of hydrophobicity (PEO-b-PCL) are more efficiently internalized as compared to PEO-b-PLA NPs. In summary, there should be a compromise regarding protein fouling and cellular uptake as resistance to non-specific protein adsorption and enhanced cellular uptake are respectively directly and inversely related to the length of the PEO stabilizing shell.

No description available.

No description available.

京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23845号 / 薬博第852号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

The objective of this study was to determine the photothermal and photochemical tissue response to carbon nanotube inclusion in laser therapy using experimental and computational methods. In this study, we specifically considered varying types and concentrations (0.01-1 mg/ml) of carbon nanotubes (CNTs), e.g., multi-walled carbon nanotubes (MWNTs), single-walled carbon nanotubes (SWNTs), and single-walled carbon nanohorns (SWNHs). In order to determine the photothermal effect of CNT inclusion, the thermal conductivity and optical properties of tissue representative phantoms with CNT inclusion were measured. Thermal conductivity of tissue phantoms containing CNTs was measured using the hot wire probe method. For identical CNT concentrations, phantoms containing MWNTs had the highest thermal conductivity. Optical properties (absorption and reduced scattering coefficients) of solutions and tissue phantoms containing carbon nanotubes were measured with spectrophotometry and determined by the inverse adding doubling (IAD) method. Inclusion of CNTs in phantoms increased light absorption with minimal effect on scattering and anisotropy. Light absorption of MWNTs was found to be higher than SWNTs and SWNHs. The photochemical response to laser irradiation (wavelength 1064 nm) of CNTs was measured with spin-trap electron paramagnetic resonance (EPR) spectroscopy. Only SWNHs appeared to produce significant levels of ROS production in response to laser excitation in the presence of NADH. We detected the predominant presence of trapped hydroxyl radical (•OH) with a trace of the trapped super oxide (O2•-) radical. These free radicals are highly reactive and could be utilized to cause targeted toxicity to cancer cells. The distribution of CNTs at the cellular level, in phantoms, and in kidney tumors was measured using transmission electron microscopy (TEM) imaging. Samples were imaged following various time periods (2-48h) of incubation and CNTs were observed inside the cell cytoplasm, nucleus, vacuole, and outside cells for the above mentioned time periods. CNTs in phantoms and tumor tissue were randomly and uniformly distributed in the entire volume. Computational model geometries were developed based on CNTs distribution in cells, tissue phantoms, and kidney tumor tissue. In the computational part of this research the temperature response to laser irradiation alone or with CNT inclusion was determined using Penne's bioheat equation which was solved by finite element methods. Experimentally measured thermal conductivity and absorption and reduced scattering coefficients were used as input parameters in Penne's bioheat equation. The accuracy of the model predicted temperature distribution was determined by comparing it to experimentally measured temperature in tissue phantoms and kidney tumors following CNT inclusion and laser therapy. The model determined temperature distribution was in close correspondence with the experimentally measured temperature. Our computational model can predict the effectiveness of laser cancer therapy by predicting the transient temperature distribution. / Ph. D.

Cancer therapies are often limited by bulk and cellular barriers to transport. Nanoparticle or chemotherapeutic compound intracellular transport has implications in understanding therapeutic effect and toxicity. The scope of this thesis was to study the intracellular transport of carbon nanohorns and to improve the efficacy of various chemotherapeutic agents through increased intracellular transport. In the first study, fluorescent probes (quantum dots) were conjugated to carbon nanohorns to facilitate the optical visualization of the nanohorns. These hybrid particles were characterized with transmission electron microscopy, electron dispersive spectroscopy and UV-VIS/FL spectroscopy. Their cellular uptake kinetics, uptake efficiencies, and intracellular distribution were determined in three malignant cell lines (breast – MDA-MB-231, bladder – AY-27, and brain – U87-MG) using flow cytometry and confocal microscopy. Intracellular distribution did not vary greatly between cell lines; however, the uptake kinetics and efficiencies were highly dependent on cell morphology. In the second study, the efficacy of various chemotherapeutic agents (i.e., doxorubicin, cisplatin, and carboplatin) was evaluated in AY-27 rat bladder transitional cell carcinoma cells. In the future, severe hyperthermia and chemothermotherapy (chemotherapy + hyperthermia) will also be evaluated. Doxorubicin and cisplatin compounds were more toxic compared to carboplatin. Hyperthermia has previously shown to increase the cellular uptake of chemotherapeutic agents; therefore, chemothermotherapy is expected to have synergistic effects on cell death. This work can then be translated to carbon nanohorn-based laser heating to generate thermal energy in a local region for delivery of high concentrations of chemotherapeutic agents. Although these two concepts are small pieces of the overall scope of nanoparticle-based therapies, they are fundamental to the advancement of such therapies. / Master of Science

There has been much interest in the use of therapeutics based on ribonucleic acid interference(RNAi) to inhibit synthesis of mutant proteins ever since Elbashir et al. (Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T., 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411, 494-498.) found that synthetic double stranded small interfering ribonucleic acids (siRNAs) can initiate this evolutionarily conserved process in mammalian cells. Since RNAi is able to target single genes and therefore mitigate the underlying molecular pathology of diseases, RNAi-based therapeutics will most likely benefit monogenic neurodegenerative diseases such as Huntington’s disease. It is however particularly difficult to deliver exogenous materials such as siRNAs into neurons in vivo as the blood-brain barrier (BBB) isolates the brain from the vascular system and prevents permeation of most materials. Neurons also do not take up exogenous materials readily. Therefore, effective delivery of siRNAs into the brain remains one of the biggest challenges impeding their use as a potential neurotherapeutic. Layered double hydroxide (LDH) nanoparticles are a class of anionic clay materials that have demonstrated great potential as a DNA (deoxyribonucleic acid) delivery system for a variety of mammalian cell lines due to their unique physiochemical properties. This thesis examined the feasibility of LDH as a siRNA delivery system for cultured neurons and demonstrated that the delivered siRNAs are able to effectively down-regulate synthesis of a target protein with minimal toxicity. Experiments were conducted using double stranded DNAs (dsDNAs) initially, and siRNAs were then used to verify these results. It was shown that nucleic acids(dsDNAs and siRNAs) could successfully intercalate into pristine LDHs to form nucleic acid-LDH complexes that had properties suitable for use as a delivery system in mammalian cells. These studies established that LDHs and nucleic acid-LDH complexes were biocompatible with neurons isolated from embryonic day 17.5 mouse cerebral cortex, suggesting that LDH can be used for nucleic acid delivery into cultured neurons. LDHs were also shown to successfully deliver nucleic acids into a non-neural mammalian cell line (NIH 3T3 cells). Finally, this thesis demonstrated for the first time that LDHs were able to deliver siRNAs into neurons, providing encouraging preliminary evidence that sequence specific gene silencing of the Mus Musculus Deleted in Colorectal Cancer (DCC) gene had occurred. However, down-regulation of the DCC protein did not occur consistently, suggesting that further optimisation is needed to improve the efficacy of siRNA-LDH complexes to inhibit expression of target protein in neurons. In future, LDHs should be further developed as an efficient siRNA delivery system for therapeutic gene silencing in the central nervous system using a neurodegenerative disease model such as the Huntington’s disease mouse model, which closely phenocopies the human disease. This model will allow the in vivo efficacy of these nanoparticles to be tested and subsequently improved in order to deliver siRNAs locally and systematically into the brain.