Not available / text

A growing number of therapeutic compounds currently being developed by pharmaceutical companies are poorly water soluble leading to limited and/or erratic bioavailability. The rate limiting step for absorption of these compounds is dependent on the dissolution and apparent solubility. Nanoparticle formation has been exploited as a method to improve the bioavailability of these poorly water soluble active pharmaceutical ingredients (API) by increasing the dissolution rates and apparent solubilities. The influence of hydrophilic stabilizers in powder compositions prepared by the spray freezing into liquid (SFL) process using either an emulsion feed dispersion or organic co-solvent feed solutions on enhancing the wetting and dissolution properties of nanostructured aggregates containing itraconazole (ITZ). Subsequently, an in vivo pharmacokinetic study was conducted comparing the SFL processed powder to commercial Sporanox®. An ultra-rapid freezing (URF) technology has been developed to produce high surface area powders composed of solid solutions of an active pharmaceutical ingredient (API) and a polymer stabilizer. Rapid freezing technologies are known to enhance the physico-chemical properties of APIs and thus increase bioavailability. However, the effect of the different freezing geometries and rates in the URF process are unknown. Therefore, this study investigated how solvent properties and thin film geometry of the droplet affect the freezing rate and thus the physico-chemical properties of micronized danazol powders. Amorphous nanoparticles containing tacrolimus (TAC) in a solid dispersion were prepared using the Ultra-rapid Freezing (URF) process. The objective of this study was to assess the effects of combinations of polymeric stabilizers on the maximum degree and extent of supersaturation of TAC. An attempt to establish if an in vitro-in vivo correlation exists between supersaturation and improved pharmacokinetic parameters for orally dosed TAC was performed. Enteric solid dispersions could overcome limitations of premature precipitation of supersaturated solutions by 1.) delaying dissolution until the compound enters the intestines where absorption is favored and 2.) increasing the apparent solubility at higher pH to increase the driving force for absorption. The objective of the study is to investigate the influence of composition parameters including drug:polymer ratio and polymer type, and particle structure of enteric solid dispersions on the release of ITZ. / text

Liposomal nanocarriers offer much promise in chemotherapeutic drug delivery because they may be specifically targeted to tumors thereby shielding healthy organs from toxic side effects of incorporated drugs. Passive targeting of liposomes is achieved through the inclusion of PEG to evade the RES and prolong circulation in the bloodstream. Since tumor vasculature exhibits increased permeability, prolonged circulation results in passive accumulation of liposomes to tumor. Active targeting is accomplished through the inclusion of agents targeted to over-expressed receptors on tumor cells. In vitro studies have demonstrated increased cytotoxicity of actively targeted liposomes due to specific uptake by tumor cells. In vivo, however, actively targeted liposomal nanocarriers have failed to meet the expectations established by the promising outcomes of in vitro studies. This is attributed to the fact that the inclusion of targeting agents results in accelerated clearance from the bloodstream and reductions in passive targeting to tumor thereby offsetting the benefits of active targeting. The central focus of this thesis was to engineer a multi-functional nanoscale drug delivery system which would enable active targeting without compromising RES evasion and passive accumulation to tumor. It was shown that the use of folate in liposomal formulations significantly reduced blood circulation times. To prevent RES recognition of folate on targeted liposomal formulations, a cysteine cleavable phospholipid-PEG conjugate was utilized to "mask" targeting ligands while liposomes were in circulation. Once passive accumulation at the tumor was achieved, cysteine was administered to detach PEG chains, expose folate, and promote uptake by tumor cells. In vivo studies demonstrated that cleavable DSPE-PEG5000 was capable of concealing folate on liposomes to maintain prolonged circulation times. In vitro studies verified the ability to conceal and expose folate on demand, permitting receptor mediated targeting and delivery of drug to target cells. Studies conducted to analyze drug uptake by tumor cells in vivo confirmed that delivery was enhanced when tumor-inoculated animals received targeted liposomes containing cleavable PEG chains followed by a cysteine infusion to expose folate. These results indicate that detachable PEG chains can be used in targeted liposomal formulations to enhance efficacy of chemotherapy in the treatment of glioma.

Histone deacetylase (HDAC) inhibition is an emerging novel therapeutic strategy in cancer therapy. HDAC inhibitors (HDACi) have shown ability to block angiogenesis and cell cycling, as well as initiate differentiation and apoptosis. In fact, suberoylanilide hydroxamic acid (SAHA) is the first in the class of HDACi approved by the FDA for the treatment of cutaneous T cell lymphoma. On the other hand, there is a sustained interest in the use of gold nanoparticles (AuNPs) for various cancer diagnostic and therapeutic applications - bioimaging, drug delivery, and binary therapy techniques such as photodynamic and photothermal therapies. This interest in AuNPs is facilitated by favorable attributes such as ease of fabrication, bioconjugation and biocompatibility, and unique optical and electronic properties. However, HDACi- and AuNPs- based antitumor agents are plagued with problems common to all chemotherapeutic agents such as lack of selectivity, which often results in systemic toxicity. Therefore, availability of a methodology to selectively deliver AuNPs and HDACi to cancer cells will significantly improve their therapeutic indices and lead to the identification of novel agents for use in diagnostic imaging and targeted cancer therapy applications.

Colloidal particles serve as useful building blocks for materials applications ranging from controlled band-gap materials to rationally designed drug delivery systems. Thus, developing approaches to direct the assembly and disassembly of sub-micron sized particles will be paramount to further advances in materials science engineering. This project focuses on using programmable and reversible binding between oligonucleotide strands to assemble and then disassemble polystyrene colloidal particles. It is shown that DNA-mediated assembly can be reversed at a fixed temperature using secondary oligonucleotide strands to competitively displace the primary strands linking particles together. It was found that 1) titrating the surface density of hybridizing probe strands and 2) adjusting the base length difference between primary and secondary target strands was key to successful isothermal disassembly. In order to titrate the surface density of primary probe-target duplexes, colloidal particles were conjugated with mixtures of probe strands and "diluent" strands in order to minimize the number of DNA linkages between particles. To reduce the steric interference of the diluent strands to hybridization events, diluent strands were clipped with a restriction enzyme in select cases. Kinetics studies revealed that a four to six base-length difference between primary and secondary target strands resulted in extensive competitive hybridization at secondary oligonucleotide concentrations as low as 10 nM. Importantly, it was found that the timing for release of either DNA alone or DNA-conjugated nanoparticles could be tuned through choices in the DNA sequences and concentration. Lastly, competitive hybridization was explored in select studies to drive the "shedding" of PEGylated DNA targets from microspheres to reveal underlying adhesive groups or ligands on the particle surface. Unlike prior work relying on elevated temperatures to melt DNA-linkages, this work presents an important first step towards extending DNA as a reversible assembly tool for physiological applications such as multifunctional drug delivery vehicles programmed to disassemble at targeted tissue sites such as malignant tumors.

Thesis (Ph.D.)--University of South Florida, 2005. / Includes vita. Includes bibliographical references (leaves 221-265).

There are currently two types of intra-arterial drug-delivery system (DDS) in clinical use in the palliative treatment of primary liver cancer. The chemotherapeutic drug doxorubicin (DOX) can be formulated into a drug-in-lipiodol emulsion (LIPDOX) or a microparticulate drug-eluting bead system (DEBDOX). To facilitate development of future DDSs, we need to understand the release and local distribution of drug from these DDSs into the complex, in vivo, pathological environment. The overall aim of this project was to assess and improve understanding of the in vivo release of DOX from LIPDOX and DEBDOX and its local disposition in the liver. These processes were investigated in detail in a multisampling-site, healthy pig model and in human patients with liver cancer. The mechanisms involved in DOX disposition were studied by examining potential interactions between DOX and lipiodol and/or cyclosporine A (CsA) in pigs.   In this project, the main elimination pathway for DOX and its primary metabolite doxorubicinol (DOXol) was via bile; their extensive canalicular carrier-mediated transport (e.g. ATP-binding cassette transporters ABCB1, ABCC1, ABCC2 and ABCG2) was inhibited by CsA. CsA had no effect on the carbonyl and aldo-keto reductases responsible for the metabolism of DOX into DOXol. LIPDOX released DOX more rapidly and to a greater extent into the circulation than DEBDOX, which had only released 15% of the dose in patients after 24 hrs. The systemic exposure to DOX was lower for DEBDOX than for LIPDOX. Greater fractions of DOXol were formed in blood and bile with LIPDOX than with DEBDOX. This may have been because DOX was more widely distributed into regions with increased metabolic capacity or because of increased intracellular uptake when DOX was delivered in LIPDOX. The excipient lipiodol in the LIPDOX formulation did not interact with transporters, enzymes or membranes that would explain the increased cellular uptake of DOX. In conclusion, the release of DOX from DEBDOX is more controlled in vivo than that from LIPDOX, indicating that DEBDOX is a more robust pharmaceutical product. The formulations for future optimized DDSs should therefore be more similar to DEBDOX than to LIPDOX.

Orientador: Beatriz Stringhetti Ferreira Cury / Coorientador: Maria Palmira Daflon Gremião / Banca: Bruno Felipe Carmelino Sarmento / Banca: Priscyla Daniely Marcato Gaspari / Banca: Marcel Otavio Cerri / Banca: Marlus Chorilli / Resumo: A GG e a P são materiais promissores para o desenvolvimento de sistemas de liberação cólon-específica de fármacos, uma vez que são resistentes às enzimas encontradas nas porções superiores do TGI, porém, são degradadas especificamente por enzimas produzidas pela microbiota colônica. Além disso, ambas apresentam mucoadesividade, o que permite a adesão do sistema ao muco, uma potencial estratégia para aumentar o tempo de retenção do fármaco no sítio alvo, bem como estabelecer um contato mais íntimo com a mucosa, ambos fatores que podem potencializar o efeito terapêutico. O resveratrol (RES) é um polifenol com reconhecidas propriedades antioxidantes e tem demonstrado outras atividades como prevenir ou retardar o início de tumores e inflamações, características promissoras para o tratamento de patologias que acometem a porção distal do TGI, como câncer colônico e colite. No entanto, por ser rapidamente absorvido e metabolizado nas porções superiores do TGI, a quantidade de RES que atinge o cólon é insuficiente para promover o efeito terapêutico desejado. As microesferas são sistemas multiparticulados, que apresentam algumas vantagens como a proteção do fármaco encapsulado e a vetorização para um sítio específico e foram empregadas na encapsulação do RES. Sua obtenção é possível através da geleificação ionotrópica, uma técnica simples, que não utiliza solventes orgânicos e com possibilidade de produção em escala industrial. O objetivo do presente estudo foi a obtenção e a caracter... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: GG and P are promising materials to be used in the development of colon specific drug delivery systems, since they are resistant to enzymes found in the upper GIT, however, they are specifically degraded by enzymes produced by colonic microbiota. In addition, both present mucoadhesiveness, which allows the adhesion of the system to the mucus, a promising strategy to prolong the time that the system remains at the target site as well as to establish a more intimate contact with the mucosa, both factors that may potentiate the therapeutic effect. Resveratrol (RES) is a polyphenol with recognized antioxidant properties and has shown other activities, such as prevent or delay the onset of tumors and inflammation, promising characteristics for the treatment of diseases that affect the distal portion of the GIT, such as colonic cancer and colitis. However, it is rapidly absorbed and metabolized in the upper portions of the GIT, the amount that reaches the colon is insufficient to promote the desired effects. Ms are a multiparticulate system that presents some advantages, such as protection of the encapsulated drug and targeting to a specific site, and were used to encapsulate RES. It is possible to prepare Ms through ionotropic gelation, a simple technique that does not use organic solvents and with the possibility of industrial scale production. The aim of the present study was to prepare dual-crosslinked Ms of GG:P through ionotropic gelation and to characterize them, intending the colon-specific release of RES. Aluminium chloride (AlCl3) and glutaraldehyde (Glu) were used as ionic and covalent crosslinkers, respectively. A saturated factorial design... (Complete abstract click electronic access below) / Doutor

The inflammatory arthritis Ankylosing Spondylitis (AS) is linked to the human leucocyte antigen HLA-B27. HLA-B27 is thought to drive AS because it misfolds during assembly in the endoplasmic reticulum (ER), inducing ER cell stress. Modulating HLA-B27 folding in the ER is therefore a therapeutic target pathway. The recent discovery of polymorphisms in the ER-resident peptidase ERAP1 that can impact on HLA-B27 and AS, makes ERAP1 one such target. Exosomes are small, typically 50-200 nm sized particles, formed in the endosomal recycling pathway, which can be released into the extracellular environment. Exosomes have a wide range of biological activities depending on the cell type of origin, and on the delivered cargo, which can include bio-active proteins, lipids, mRNA and miRNA. There is interest in the use of exosomes as drug delivery agents. Here, exosomes were studied as a delivery agent to modulate ERAP1, as a potential therapeutic tool for the treatment of AS. Exosomes, isolated from cell lines including CEM and Jurkat (T cell lineage), Jesthom (B cell lineage), U937 (monocyte lineage) and the epithelial HeLa cell line, were characterized by nanoparticle tracking analysis, flow cytometry and immunoblotting using markers including CD9, CD63, CD81 and TSG101. Differential expression of these markers in the immune cell lines indicated the complexity of defining exosomes. EVs were then tested using cell penetrating peptides, electroporation, lipid transfection and sonication for their ability to load FITC-siRNA or FITC-antibody as cargo. Significantly, post-loading RNase A or trypsin incubation demonstrated that many techniques do not lead to efficient cargo loading of exosomes. Sonication proved the most effective technique, with up to 30% efficiency. Loading of exosomes with ERAP1-targetted siRNA did not however lead to notable ERAP1 inhibition. The data indicates that external loading of exosomes with cargo remains a significant challenge in developing exosomes as therapeutic tools.

Orientador: Prof. Dr. Fernando Carlos Giacomelli / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2017. / O uso de nanopartículas poliméricas vem progressivamente se intensificando nos últimos anos, principalmente em que pese a aplicações na área biomédica. Neste cenário, o foco principal reside na utilização de polímeros biodegradáveis para fabricação de sistemas nanocarregadores de agentes ativos. Particularmente no campo da nanomedicina, a habilidade de se controlar a dimensão de nanopartículas, bem como se entender o perfil de liberação de pequenas moléculas encapsuladas é essencial. Levando-se em conta estas considerações, o objetivo deste trabalho foi fabricar nanopartículas poliméricas biodegradáveis de PCL e PLGA carregadas com a sonda cumarina-6 e analisar as diversas variáveis de formulação a fim de se entender a influência das características estruturais das nanopartículas e a sua relação com o teor encapsulado, eficiência de encapsulação, perfil de liberação e captura celular dos sistemas nanoestruturados. Os sistemas coloidais produzidos foram caracterizados por meio de técnicas de espalhamento de luz dinâmico (DLS), estático (SLS) e eletroforético (ELS) e espectroscopia de fluorescência. A caracterização estrutural detalhada das formulações produzidas sugere que a densidade das partículas, independente das variáveis de formulação, é substancialmente menor do que a densidade dos polímeros sólidos, implicando que as cadeias poliméricas que formam as nanopartículas estão pouco compactadas e por consequência, os sistemas supramoleculares são substancialmente hidratados (os cálculos mostram conteúdos de água formando os agregados entre 72% e 95% volume/volume). As medidas de espectroscopia de fluorescência evidenciaram que é possível produzir sistemas nanocarregadores onde a eficiência de encapsulação atinge valores maiores do que 50%, entretanto, independente das variáveis de formulação, o teor encapsulado nunca ultrapassa 0,4% massa/massa. Acreditamos que os baixos teores de sonda encapsulada estejam essencialmente relacionados a densidade das partículas e a característica de elevada hidratação. As investigações também demonstram que a liberação da sonda encapsulada é essencialmente governada pelo movimento de difusão. Consequentemente, a biodegradabilidade dos poliésteres utilizados na produção dos coloides pode ter efeito apenas na excreção do material polimérico de ambientes biológicos, mas não parece ter efeito no processo de liberação controlada de princípios ativos encapsulados. Por final, foram feitas análises de microscopia de fluorescência e citometria de fluxo para avaliar a influência dos parâmetros estruturais na captura e internalização celular dos sistemas poliméricos nanoestruturados. Os dados mostram que nanopartículas produzidas a partir de tetraidrofurano (THF), portanto maiores parecem ser internalizadas de maneira mais eficiente pelo menos na região de tamanhos investigada. Os dados também mostram serem inconclusivas as influências da hidrofobicidade e carga superficial dos sistemas. / The uses of polymeric nanoparticles came intensifying for a period of time, and are being applicable in the biomedical area. In this scenario, the focus is use biodegradable polymers to production of nanocarrier systems of active agents. Particularly in the field of nanomedicine, the ability to control the size of nanoparticles, as well as understanding the release profile of small-encapsulated molecules, is essential. Taking into account these considerations, the objective of this work was to manufacture biodegradable polymeric nanoparticles of PCL and PLGA loaded with the coumarin-6 probe and to analyze the various formulation variables in order to understand the influence of a structural characteristics of nanoparticles, and their relation with the encapsulated content, encapsulation efficiency, release profile and cellular capture of nanostructured systems. The colloidal systems were produced to characterizing by dynamic light scattering (DLS), static (SLS) and electrophoretic (ELS) techniques and fluorescence spectroscopy. The detailed structural characterization of formulations suggested that density of particles, regardless of a formulation variable, is substantially lower than the density of the solid polymers, implying that polymer chains forming the nanoparticles are poorly compacted and consequently the supramolecular systems are substantially hydrated (the calculations show water contents forming the aggregates between 72% and 95% v/v). The fluorescence spectroscopy measurements showed that it is possible to produce nanocarrier systems where the encapsulation efficiency reaches values higher than 50%, however, regardless of the formulation variables; the encapsulated content never exceeds 0.4% w/w. We believe that the low levels of encapsulated probe are essentially related to particle density and the characteristic of high hydration. The investigations also demonstrate that the release of a encapsulated probe is essentially governed by the diffusion movement. According to biodegradability of polyesters used in the production of colloids may have effect only in the excretion of polymer material from biological environment, but does not appear to have an effect on the controlled release process of encapsulated active principles. Finally, fluorescence microscopy and flow cytometer analyzes the performances to evaluate the influence of structural parameters on capture and cellular internalization of nanostructured polymer systems. The data shows that nanoparticles produced from tetrahydrofuran (THF), therefore larger, appear to be internalized more efficient at least in the region of investigation sizes. The data also shows that the influences of the hydrophobicity and surface charge of the systems was inconclusive.