DNA vaccines offer unique promise as a means of generating immunity against infectious and malignant disease. Unfortunately a number of obstacles, including rapid degradation of naked plasmid DNA (pDNA), poor cellular uptake by antigen presenting cells (APCs) and subsequent low levels of gene expression have limited the ability of DNA vaccines to raise sufficient immune responses towards the target antigen. This thesis is focused on investigating the immunostimulatory potential of liposomal nanoparticulate (LN) formulations of pDNA (stabilized plasmid lipid particles; SPLP) and cytosine-guanine oligodeoxynucleotides (CpG-ODN; LN CpG-ODN), and examining their ability to act together as a non-viral DNA vaccine in attempt to address the shortcomings of current DNA vaccine approaches. One focus of this thesis concerns investigating the immunostimulatory activity of LN formulations of CpG-ODN and pDNA. It is shown that despite dramatic differences in pharmacokinetics and biodistribution of LN CpG-ODN following intravenous (i.v.) and subcutaneous (s.c.) administration the resultant immune response is very similar, which is concluded to be due to the intrinsic ability of APCs to sequester LN CpG- ODN. In addition, it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA and it is observed that SPLP maintains immunostimulatory activity in Toll-like receptor 9 (TLR9) knock-out mice. Together theses findings highlight the need for DNA-based therapies to consider both TLR9-dependent and -independent immunostimulatory activities of pDNA when constructing non-viral vectors. Furthermore, a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpG-ODN is introduced. The ability of vaccination with SPLP to act prophylactically, to protect mice from tumour challenge, and therapeutically, in a novel vaccination strategy where the antigen is expressed at the tumour site as a result of SPLP-mediated transfection, is explored, demonstrating that in the presence of LN CpG-ODN SPLP possesses potential as a non-viral delivery system for DNA-based cancer vaccines. In summary, this work represents a substantial advance in the understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN and provides insight into their ability to work together as a non-viral DNA vaccine.

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DNA vaccines offer unique promise as a means of generating immunity against infectious and malignant disease. Unfortunately a number of obstacles, including rapid degradation of naked plasmid DNA (pDNA), poor cellular uptake by antigen presenting cells (APCs) and subsequent low levels of gene expression have limited the ability of DNA vaccines to raise sufficient immune responses towards the target antigen. This thesis is focused on investigating the immunostimulatory potential of liposomal nanoparticulate (LN) formulations of pDNA (stabilized plasmid lipid particles; SPLP) and cytosine-guanine oligodeoxynucleotides (CpG-ODN; LN CpG-ODN), and examining their ability to act together as a non-viral DNA vaccine in attempt to address the shortcomings of current DNA vaccine approaches. One focus of this thesis concerns investigating the immunostimulatory activity of LN formulations of CpG-ODN and pDNA. It is shown that despite dramatic differences in pharmacokinetics and biodistribution of LN CpG-ODN following intravenous (i.v.) and subcutaneous (s.c.) administration the resultant immune response is very similar, which is concluded to be due to the intrinsic ability of APCs to sequester LN CpG- ODN. In addition, it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA and it is observed that SPLP maintains immunostimulatory activity in Toll-like receptor 9 (TLR9) knock-out mice. Together theses findings highlight the need for DNA-based therapies to consider both TLR9-dependent and -independent immunostimulatory activities of pDNA when constructing non-viral vectors. Furthermore, a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpG-ODN is introduced. The ability of vaccination with SPLP to act prophylactically, to protect mice from tumour challenge, and therapeutically, in a novel vaccination strategy where the antigen is expressed at the tumour site as a result of SPLP-mediated transfection, is explored, demonstrating that in the presence of LN CpG-ODN SPLP possesses potential as a non-viral delivery system for DNA-based cancer vaccines. In summary, this work represents a substantial advance in the understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN and provides insight into their ability to work together as a non-viral DNA vaccine.

The use of multimodal imaging as a tool to assess the in vivo pharmacokinetics and biodistribution of nanoparticles is important in drug development and imaging-guided therapy. The current study reports the use of combined micro-CT and optical imaging FMT to quantitatively assess the whole body and intratumoural distribution of a nano-sized liposome-based CT/optical imaging probe carrying iohexol and Cy5.5. Micro-CT was used to guide the FMT tumour delineation and signal correction. This investigation demonstrates the critical role micro-CT can play in guiding FMT-based quantification of distribution. As well the combination of CT and optical imaging enable visualization of the liposomes at the whole body, tumor and cellular levels with high sensitivity and excellent anatomical background. Such non-invasive assessment of therapeutic distribution at the macro and micro scale is necessary for implementation of personalized medicine including image-guided patient stratification and real-time adjustment of therapeutic dose.

The use of multimodal imaging as a tool to assess the in vivo pharmacokinetics and biodistribution of nanoparticles is important in drug development and imaging-guided therapy. The current study reports the use of combined micro-CT and optical imaging FMT to quantitatively assess the whole body and intratumoural distribution of a nano-sized liposome-based CT/optical imaging probe carrying iohexol and Cy5.5. Micro-CT was used to guide the FMT tumour delineation and signal correction. This investigation demonstrates the critical role micro-CT can play in guiding FMT-based quantification of distribution. As well the combination of CT and optical imaging enable visualization of the liposomes at the whole body, tumor and cellular levels with high sensitivity and excellent anatomical background. Such non-invasive assessment of therapeutic distribution at the macro and micro scale is necessary for implementation of personalized medicine including image-guided patient stratification and real-time adjustment of therapeutic dose.

J E. coli On-Off &ldquo, &rdquo, °, &ndash The specific objectives of the work presented in this dissertation are to design novel molecular sensors based on fluorescence resonance energy transfer (FRET) between fluorophore (donor) and polydiacetylene (PDA, acceptor) for selective detection of biomolecules in solution. The work described in this dissertation is divided into three sections. In the first section, we report here two novel systems where the rate of energy transfer is based on changes in the spectral overlap between the emission of the donor and the absorption of the acceptor (J) as well as changes in the quantum yield of the acceptor. In the second section, we discuss modified these high sensitive molecular sensors based on FRET by using different receptors for selective detection of biomolecules such as proteins or bacteria in solution. The third section develops reversibility studies on FRET based sensors in solution or solid state. In the Chapters two and three, conjugated polydiacetylene (PDA) possessing stimuli-responsive properties have been intensively investigated for developing efficient sensors. Sensors based on FRET between conjugated polymers and fluorophores can be more sensitive than colorimetric based sensors. We use the fluorophore dansyl as the donor and polydiacetylene (PDA) as the acceptor to demonstrate the modulation of FRET efficiency through conformationally induced changes in the PDA absorption spectrum following thermal treatment that converts the PDA backbone of the liposome from the blue form to the red form. We have used steady-state electronic absorption, emission and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). Energy transfer was found to be significantly more efficient from dansyl to the red-form PDA. This is due to large increase in the J values between dansyl emission and absorption red-form of PDA. We also have found that the monomer ratio of acceptor to donor (Rad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. A decrease in Rad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. Increase in Rad led to increase probability of FRET from donor to acceptor as larger number of acceptors are present around a given donor. The competition between donor for energy transfer increases with decrease in Rad that contributed to lower FRET efficiency between donors and acceptors. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to the insertion of dansyl in the bilayer of the liposomes which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. Much larger emission amplification for FRET was observed as compared to direct-excitation of PDA. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. These findings support the basis of a new sensing platform that utilizes J-modulated FRET as an actuating mechanism. A FRET based protein sensor by using sulforhodamine 101 as donor and PDA as acceptors was developed. This novel FRET based system primarily utilizes changes in J values (the spectral overlap between the emission of the donor and absorption of the acceptor) for the modulation of FRET efficiency between donors and acceptors. These FRET based sensors can be modified by tagged receptors (for proteins, viruses, and bactria) onto PDA liposomes which can interact with ligands present on proteins or bacteria. The biotin-streptavidin interactions were used as a sensing model system to test our FRET sensor response. In chapter 4, four different biotin-tagged lipids were used as receptors to investigate the effect of interactions between ligand-receptors on the FRET efficiency. The biotin was covalently linked to the liposome surface when using biotin-tagged diacetylene; whereas the biotin-tagged lipids with hydrophobic chains but without diacetylene functionalities provided non-covalently inserted lipids in liposomes. These studies were used to elucidate the effect of molecular interactions on FRET sensor response. The conjugated polymerized liposomes consisted of sulforhodamine-tagged-diacetylene and receptors linked lipids in different molar ratios. The characterization of the liposomes and sensing mechanism was investigated using UV-Vis and steady-state emission spectroscopy. The liposome solution yielded a weak donor emission (sulforhodamine 101) from after photo-polymerization of diacetylene monomers. This is due to energy transfer from the donor to PDA backbone chains (acceptors). The addition of streptavidin which interacted with biotin receptors resulted in increase in the sulforhodamine 101 emissions. The stress, due to interactions between biotin and streptavidin, induced the chromatic shift in the absorption spectrum of PDA which led to a decrease in the spectral overlap (J) between the emission spectra of donor and the absorption spectra of acceptor, leading to a decrease in the FRET efficiency from sulforhodamine 101 to PDA. These sensors, thus, show an "On-Off" type optical mechanism based on FRET between fluorophores and PDA where the donor emission was highly quenched in the "Off" state but was turned "On" due to receptor-ligand interactions. Large electronic absorbance and emission intensity differences between covalently and non-covalently bound biotin liposome systems were observed which indicated that the molecular interactions between biotin and PDA backbone play a crucial role in the FRET sensor response. In Chapter 5, we also developed FRET sensor for the detection of E. coli in aqueous media. Two glucose-based receptors were used in this study: (1) glucose-tagged lipid which can be inserted non-covalently in the bilayer of liposome, and (2) glucose-tagged diacetylene monomer in which the receptors were covalently bound to the backbone of the PDA liposome. The steady-state UV/Vis absorbance and fluorescence emission spectroscopy, and the fluorescence microscopy analysis of the receptors-containing liposomes were investigated for the detection of E. coli. The blue shift in the absorption spectrum of the conjugated PDA backbone induced through the interactions between receptors and bacteria resulted in decrease in the spectral overlap between the emission of SR-101 (donor) and the absorption of PDA (acceptor). This, ultimately, led to change in FRET efficiency between SR-101 and PDA after glucose - E. coli binding and caused increase in the emission intensity of SR-101. Polydiacetylenes have been exploited because of their sensitivity to external stimuli, such as temperature, pH, ions, and ligands. Unfortunately, the majorities of the sensors developed are not reversible but used as a one-time use. Here we report our preliminary results of a benzoic acid monomer of polydiacetylene (PDA-mBzA) to investigate reversible FRET characteristics between fluorophore and PDA. The LS films containing dansyl-tagged-diacetylene monomers and m-aminobenzoic acid derivatized- diacetylene monomers in different molar ratios were self-assembled and polymerized. The UV/Vis and steady-state fluorescence emission analysis of these LS films were investigated. These systems have shown partial reversible FRET over many "on-off" cycles. We believe that this incomplete FRET reversibility is due to liposomes preparation conditions used for liposomes which decreased PDA-mBzA amount in liposomes. We also reported reversible FRET studies on the liposome solutions, made from the monomer of m-aminobenzoic acid derivatized-10,12-pentacosadiynoic acid (PDA-mBzA) monomers and 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA) or dansyl-tagged diacetylene. After photo-polymerization, the solution appeared blue in color at room temperature. Heating and cooling cycles (between 25 ºC and 95 ºC temperature range), illustrated a visible color change from blue to red and a complete return to blue over many thermal cycles. Our preliminary reversible absorption and emission measurements showed that there exist opportunities for reversibility in FRET response. We are now performing more experiments to increase the FRET reversibility in these experiments. Although our system does not display full reversibility, the preliminary absorption and emission measurements strongly suggest that there exist opportunities for fully reversible selective and sensitive FRET-based sensors after further optimization of the system.

The main aim of work presented here is to design, develop and characterize a colorimetric model membrane (liposome) systems, which can bind with proteins, enzymes, bacteria, virus and other biomolecules. PDA molecules are utilized as a scaffold for the bilayer membrane, and a colorimetric assay is carried out. The holy grail of present work contributes towards the better understanding of protein interactions with the cell bilayer surface. Chapter 1 introduces a brief history on the advent of bilayer systems for cellular research exploration. We presented a literature survey about how liposome systems are used as a complementary technique to understand the fundamental principles of cellular membrane functions. Furthermore, we describe about membrane protein functions and recent findings on how proteins interact with the cell membrane. Finally, we explain conjugated systems and their exploration in bilayer membrane as a colorimetric scaffold. We also touch bases with major fluorescence techniques used in our experiments. Chapter 2 provides details on the preparation protocols of liposome and liposome-protein complexes. We confirmed protein-bilayer interactions by monitoring FRET between PDA and rhodamine molecules. Furthermore, we performed streptavidin-biotin binding studies on the PDA bilayer. Protein binding changed the spectral overlap (J) between PDA and rhodamine, which ultimately increased the fluorescence emission of rhodamine. The goal of performing these studies was to present a complete protocol for the preparation of liposome and protein-liposome complex. In chapter 3, we investigate how proteins bind on the cell membrane. Additionally, we propose a model of protein-bilayer complex. We reported that, by harnessing cell bilayer with specific bio-molecules, we monitored protein--bilayer, protein--protein and enzyme--substrate signal transduction. We have developed a colorimetric system for monitoring vital stimulations occur on the liposomal membrane surface. Bilayer was modified to covalently bind the amino group of lysine residues present on protein molecules. These bio-molecular interactions on bilayer surface provide differential stimulus, which turned out to be the major cause of differential spectroscopic signals depending upon size and shape of the protein bounded to the bilayer. Polydiacetylene (PDA) liposomes are the core of our color based system. These liposomes are used to monitor subtle interactions on the bilayer surface. We have also developed a semi-quantitative method based on the colorimetric response of PDA liposomes; we were able to detect protein molecules at sub-nanomolar concentrations in the solution. It's capability of distinguishing protein molecules based on their chemical and physical interactions to bilayer contributes towards the identity of our system. Interestingly, our mass spectroscopic data suggested non-specific enzymatic cleavage of membrane-bound proteins. These fragments were not present in bulk protein cleavage. We also proposed a model that depicts the covalent binding of protein at the bilayer of liposomes. These studies are intended to investigate protein-bilayer and enzyme-protein interaction occurring on the cell surface. In chapter 4, we focus on the kinetics of protein interaction on bilayer surface and we also attempt to visualize these interactions by exploring fluorescence microscopy. A self-assembled cell membrane is consisted of various lipids, which cluster themselves in their preferred phase separated regions. Lipid clusters are very important for lipid specific protein interactions. We investigated protein binding on such phase separated regions under a fluorescence microscope. Furthermore, we enzymatically catalyzed proteins, which were covalently bonded on the bilayer surface. This catalytic reaction was monitored both spectroscopically and under a fluorescence microscope. These studies were performed to help us in the better understanding of biological interactions at cell surface. Chapter 5, describes the encapsulation and controlled delivery of antimicrobial compounds from liposomes. Use of antimicrobial coatings on food packaging is one of the important technologies of active packaging for improving food safety. There is growing demand for natural antimicrobials because of fear of adverse health effects of synthetic preservatives. The main objective of this study is to compare antimicrobial activity of free versus encapsulated curcumin. Glass surfaces coated with nano-encapsulated curcumin may be used as an active packaging material in preserving liquid foods; however, further study is required to improve antimicrobial activities of polylactic acid PLA surfaces. In chapter 6, we investigate interactions between receptors and ligands at bilayer surface of polydiacetylene (PDA) liposomal nanoparticles using changes in electronic absorption spectroscopy and fluorescence resonance energy transfer (FRET). We study the effect of mode of linkage (covalent versus noncovalent) between the receptor and liposome bilayer. We also examine the effect of size-dependent interactions between liposome and analyte through electronic absorption and FRET responses. Glucose (receptor) molecules were either covalently or noncovalently attached at the bilayer of nanoparticles, and they provided selectivity for molecular interactions between glucose and glycoprotein ligands of E. coli. These interactions induced stress on conjugated PDA chain which resulted in changes (blue to red) in the absorption spectrum of PDA. The changes in electronic absorbance also led to changes in FRET efficiency between conjugated PDA chains (acceptor) and fluorophores (Sulphorhodamine-101) (donor) attached to the bilayer surface. Interestingly, we did not find significant differences in UV−Vis and FRET responses for covalently and noncovalently bound glucose to liposomes following their interactions with E. coli. We attributed these results to close proximity of glucose receptor molecules to the liposome bilayer surface such that induced stress were similar in both the cases. We also found that PDA emission from direct excitation mechanism was ∼2−10 times larger than that of the FRET-based response. These differences in emission signals were attributed to three major reasons: nonspecific interactions between E. coli and liposomes, size differences between analyte and liposomes, and a much higher PDA concentration with respect to sulforhodamine (SR-101). We have proposed a model to explain our experimental observations. Our fundamental studies reported here will help in enhancing our knowledge regarding interactions involved between soft particles at molecular levels. In chapter 7, we conclude the summary of all work carried out in previous chapters.

Made available in DSpace on 2014-06-11T19:24:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-08-04Bitstream added on 2014-06-13T19:48:18Z : No. of bitstreams: 1 oliveira_lc_me_arafcf.pdf: 1008043 bytes, checksum: 986417155d3a5f5fe5fe070125ff8109 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Neste trabalho estudou-se a encapsulação do cloridrato de ciprofloxacino (CIPRO) em lipossomas revestidos de ácido poli-láctico-co-glicólico (PLGA) ou o copolímero termosensível Pluronic® F127 (PLU). A eficiência de encapsulação foi obtida a partir das frações contendo lipossomas carregados de CIPRO separadas por cromatografia de exclusão em gel de Sephadex G-50, e mostrou-se melhor para as amostras contendo PLGA e maior concentração de CIPRO (5 mg/mL). A determinação do diâmetro médio dos lipossomas foi realizada por espalhamento dinâmico de luz (Light Scattering) e demonstrou redução no tamanho das estruturas quando PLGA ou PLU estavam presentes nas preparações. A incorporação de CIPRO aos lipossomas provocou aumento do tamanho das estruturas quando comparadas com as preparações isentas de fármaco. Verificou-se que o aumento na concentração de fármaco provocou a diminuição do diâmetro médio dos lipossomas. Experimentos de liberação in vitro mostraram que a liberação do CIPRO a partir dos lipossomas foi mais lenta em relação ao CIPRO não encapsulado. A liberação do CIPRO a partir de lipossomas revestidos com PLGA mostrou que a liberação foi mais lenta em relação aos lipossomas não revestidos. Os resultados demonstram que as preparações de CIPRO em lipossomas revestidos com PLGA ou PLU podem representar sistemas de liberação de fármacos antibióticos com grande potencial de utilização. O estudo de biodisponibilidade ocular demonstrou que lipossomas revestidos com PLGA e PLU mantiveram a MIC90 de CIPRO para os principais patógenos oculares por mais tempo que o CIPRO em solução no humor aquoso, quando administrados por via subconjuntival. Estes resultados demonstram que a associação de PLGA e PLU com lipossomas pode ser utilizada como um eficiente sistema de liberação ocular de fármacos. / In the present work we studied the encapsulation of ciprofloxacin hydrochloride (CIPRO) in liposomes coated either by the poly-lactic-co-glycolic acid (PLGA) or thermosensitive copolymer Pluronic® F127 (PLU). Unilamellar liposomes containing 40 or 50 mM of hydrogenated soy phosphatidylcholine (FSH) were prepared by reverse phase evaporation (REV) method, followed by sonication. Encapsulation efficiency was obtained using fractions containing liposomes loaded with CIPRO, separated by exclusion chromatography on Sephadex G-50 gel. Best encapsulation efficiency was obtained with samples containing PLGA with CIPRO at a higher concentration (5 mg/mL). Liposome medium diameter was determined by dynamic light scattering, and showed a size reduction when either PLGA or PLU was present in the preparations. The incorporation of CIPRO into the liposomes caused a size increasement of the structures when compared to preparations lacking the drug. Nevertheless, increasing the drug concentration caused a decrease of the liposome medium diameter. Experiments of in vitro release showed that the liberation of CIPRO from the liposomes was slower when compared to not encapsulated CIPRO. The release of CIPRO from liposomes coated by PLGA showed that the liberation was slower when compared to non-coated liposomes. The results show that liposome preparations containing CIPRO and covered either by PLGA or PLU represent antibiotic drug delivery systems with great possibilities. The bioavailability study shows that liposomes covered by both PLGA and PLU maintained the MIC90 of CIPRO against the main ocular pathogens for longer time than CIPRO solution in aqueous humor, when subconjunctivally injected. These results demonstrate that the PLGA and PLU association with liposomes can be used as an efficient ocular drug delivery system.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17541号 / 工博第3700号 / 新制||工||1563(附属図書館) / 30307 / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 清水 芳久, 教授 田中 宏明, 教授 米田 稔 / 学位規則第4条第1項該当