Despite the great success of hip implant surgeries, wear particle-induced implant aseptic loosening still limits the implant longevity. Simvastatin, an FDA-approved cholesterol lowering statin, is a promising drug candidate for the treatment of implant aseptic loosening due to its anti-inflammatory properties as well as its ability to stimulate bone growth and inhibit bone resorption. In addition, alginate microspheres have been used extensively in drug delivery applications because of alginate properties, including biocompatibility and gelation in mild conditions. However, the hydrophobicity of simvastatin, as well as the large alginate microsphere pore size leading to the leakage of low molecular weight drugs are limiting factors for their use as a delivery system for simvastatin. Therefore, the objectives of this thesis were twofold: 1. To complex simvastatin with 2-hydroxypropyl-β-cyclodextrin (HP-βCD) in order to increase its solubility; and 2. To increase simvastatin encapsulation efficiency in alginate microspheres by coating the microspheres with chitosan, adding dextran sulfate in the alginate solution, and optimizing the gelation conditions used for the synthesis of the microspheres (e.g., volume of gelation medium, curing time, and addition of simvastatin in the gelation medium). Results showed that simvastatin complexation with HP-βCD increased with HP-βCD to simvastatin molar ratio, to a maximum of 97.6% at the molar ratio of 10. Results also showed that chitosan coating of the alginate microspheres increased simvastatin encapsulation efficiency (up to 10.6%), which was further improved (up to 14.0%) when adding 2.0% (w/v) dextran sulfate to the alginate solution. This increase was likely due to electrostatic interactions between dextran sulfate and chitosan in addition to alginate, resulting in a denser coating. Finally, the addition of simvastatin in the gelation medium was shown to also increase simvastatin encapsulation (up to 22.4%), likely because of a decrease in the diffusion of simvastatin out of the microspheres. Overall, this work completed the initial steps for the development of an alginate-based drug delivery system for simvastatin with the long-term goal of providing a local delivery of simvastatin to modulate implant aseptic loosening.

The aim of this study was to investigate in vitro transdermal diffusion of a small peptide namely arginine vasopressin (AVP) with the aid of the novel PheroidTM drug delivery system. Generally, peptides seem unfit for transdermal permeation, but it was thought prudent to explore the suitability of this lipid-based system after success was achieved with entrapment of tuberculostatics, bacteria and viruses. Bestatin (a selective aminopeptidase inhibitor) was employed to circumvent any skin-related degradation of the active. Therefore, the effect of bestatin on the preservation of AVP during diffusion was investigated. Vertical Franz cell diffusion studies were conducted with female abdominal skin, with AVP at a concentration of 150 pglml in the donor phase and Hepes buffer as the receptor phase over a twelve-hour period. To prove entrapment of AVP within the lipid structures of the PheroidsTM, fluorescentlylabelled samples were monitored by means of confocal laser scanning microscopy (CLSM), which revealed definite entrapment. In vitro permeation profiles for AVP exhibited a biphasic character, with the majority of permeation occurring during the first two hours. The PheroidTM delivery system proved to be advantageous when applied as delivery medium. The inclusion of bestatin has an enhancing effect on permeation probably due to its protection of AVP. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23161号 / 工博第4805号 / 新制||工||1751(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 田畑 泰彦, 教授 秋吉 一成, 教授 沼田 圭司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Considerando que a obtenção de uma formulação farmacêutica pode ser melhor planejada se algumas condições biológicas inerentes à cavidade oral forem contempladas e que os sistemas de liberação modificada podem ser ferramentas para o controle de doenças orais, o objetivo do trabalho é obter uma formulação contendo clorexidina, avaliando seu efeito sobre os mesmos. Este trabalho foi dividido em duas etapas, sendo que a primeira envolveu a obtenção da formulação mais adequada aos experimentos biológicos e a segunda avaliou seus efeitos sobre biofilmes patogênicos orais. Assim, formulações com diferentes concentrações de clorexidina foram preparadas e avaliadas visualmente em relação à sua integridade física nas condições de crescimento dos biofilmes, seguido de ensaio de liberação em meio estático utilizando tampão como meio de dissolução (meio de dissolução convencional). A formulação selecionada foi submetida a ensaios de liberação em meio estático contendo os diferentes caldos de cultura bacterianos como meio de dissolução. Na segunda etapa do trabalho, o efeito da formulação selecionada foi avaliado em biofilmes cariogênicos de Streptococcus mutans ou biofilmes periodontopatogênicos de Porphyromonas gingivalis. Biofilmes de S. mutans (n=6) ou P. gingivalis (n=3) foram formados em caldos de cultura sob lâminas de vidro por 6 e 3 dias, respectivamente, sendo expostos a um dos seguintes tratamentos: 1) Formulação contendo 92% de quitosana e 8% de hidroxipropil metilcelulose (CV, como controle do veículo) ou 2) Formulação contendo 82% de quitosana, 8% de hidroxipropil metilcelulose e 10% de clorexidina (CHX, grupo experimental). Um grupo sem exposição à formulação foi incluído como controle negativo (CN). Após o período experimental, a viabilidade bacteriana, o pH dos biofilmes e a quantificação de clorexidina liberada para os caldos de cultura foram determinados. Os dados foram analisados estatisticamente por teste de Tukey-Kramer e Tukey, sendo o nível de significância estabelecido em 5%. Os resultados sugerem que ainda que a liberação de clorexidina da formulação nos caldos de cultura de S. mutans e P. gingivalis tenha sido menor se comparada ao meio de dissolução convencional (p<0,05), o efeito biológico promovido foi observado para ambos os biofilmes. Para o pH dos biofilmes de S. mutans, os grupos CN e CV não apresentaram diferença entre si (p>0,05), mas apresentaram quedas de pH maiores se comparados à CHX (p<0,05). CHX também resultou em menor viabilidade bacteriana dos biofilmes se comparada aos controles (p<0,05), que não apresentaram diferença entre si (p>0,05). Para P. gingivalis também houve mais morte celular nos biofilmes expostos à CHX (p<0,05) mas CV também apresentou perda de viabilidade em comparação à CN (p<0,05). Apesar da liberação de clorexidina da formulação ter sido dificultada pela presença dos microrganismos, os resultados sugerem que o sistema de liberação obtido foi capaz de diminuir a patogenicidade dos biofimes de Streptococcus mutans e de Porphyromonas gingivalis. Assim, o presente estudo sugere a importância de aliar os estudos de diferentes áreas de conhecimento de forma a contribuir no planejamento das formulações, vislumbrando futuros benefícios clínicos para o controle das doenças orais. / Considering that obtaining a pharmaceutical formulation can be better planned if some biological conditions inherent to the oral cavity are contemplated and that the modified release systems may be tools for the control of oral diseases, the aim of the work is to obtain a formulation containing chlorhexidine and evaluate its effect in pathogenic biofilms. This work was divided in two stages, the first involved obtaining the most appropriate formulation for biological experiments and the second evaluate its effect on oral pathogenic biofilms. Thus, formulations with different concentrations of chlorhexidine were prepared and evaluated visually for their physical integrity under the biofilm growth conditions, followed by the static media release assay using buffer as the dissolution medium (conventional dissolution medium). The selected formulation was subjected to static release tests containing the different bacterial culture broths as the dissolution medium. In the second stage of the work, the effect of the selected formulation was evaluated in cariogenic biofilms of Streptococcus mutans or periodontopathogenic biofilms of Porphyromonas gingivalis. Biofilms of S. mutans (n = 6) or P. gingivalis (n = 3) were formed in culture broths under glass slides for 6 and 3 days respectively, being exposed to one of the following treatments: 1) Formulation containing 92 % of chitosan and 8% of hydroxypropyl methylcellulose (CV, vehicle control) or 2) Formulation containing 82% of chitosan, 8% of hydroxypropyl methylcellulose and 10% of chlorhexidine (CHX, experimental group). A group without exposure to the formulation was included as negative control (CN). After the experimental period, bacterial viability, biofilms pH and quantification of chlorhexidine released into the culture broths were determined. Data were statistically analyzed by Tukey-Kramer and Tukey test with a level of significance of 5%. Results suggest that although chlorhexidine release from formulation in the culture broths of S. mutans and P. gingivalis was lower compared to the conventional dissolution medium (p <0.05), the treatment promoted biological effect for both biofilms. Regarding S. mutans biofilms pH, CN and CV groups showed no difference (p> 0.05), but showed higher pH drops when compared to CHX (p <0.05). CHX also resulted in lower bacterial viability of biofilms compared to controls (p <0.05), which did not show any difference (p> 0.05). For P. gingivalis, there was higher cell death in the biofilms exposed to CHX (p <0.05) and CV presented loss of viability compared to CN (p <0.05). Although the release of chlorhexidine from the formulation has been hampered by the presence of microorganisms, results suggest that the release system was able to reduce the pathogenicity of Streptococcus mutans and Porphyromonas gingivalis biofilms. Thus, the present study suggests the importance of combining studies from different areas of knowledge in order to contribute to the design of formulations aiming future clinical benefits for the oral diseases control.

Au cours des dernières décennies, la technologie des nanoparticules pour la délivrance des vaccins au niveau de muqueuses a reçu un intérêt croissant. L’administration intranasale possède de grands avantages pour la stimulation du système immunitaire, telles que la stimulation d’une immunité protectrice locale et systémique. Cependant des systèmes de délivrance et des adjuvants sont souvent nécessaires pour déclencher efficacement la réponse immunitaire. Nous avons appliqué la technologie des nanoparticules en tant que système de délivrance d'un vaccin universel nasal contre la grippe dans un projet européen FP7 appelé UniVacFlu. Nous avons formulé un antigène adjuvé CTA1-3M2e-DD avec les NPL. Cet antigène est composé de la sous-unité A1 de la toxine du choléra et d’un épitope conservé du virus de la grippe A (M2e), ainsi que du dimère de l’analogue synthétique de la protéine A de Staphylococcus aureus (DD). Les nanoparticules utilisées sont poreuses et constituées de maltodextrines réticulées ayant un coeur lipidique (NPL). L’association de cet antigène avec les NPL est quantitative et la formulation est stable pendant au moins six mois à 4°C. Les NPL permettent également de délivrer d’une manière accrue cet antigène dans les cellules épithéliales des voies respiratoires et les macrophages. Actuellement ces formulations sont évaluées chez la souris par le consortium UniVacFlu.L'un des principaux problèmes des vaccins nasal est la toxicité qui peut être provoquée par le passage nez-cerveau de l'un de ses composants. Le but de ce travail est d'évaluer le potentiel des NPL, en tant que vecteurs pour la délivrance des vaccins nasal. Ainsi, nous avons étudié le chargement d’un antigène dans les NPL et sa délivrance dans les cellules épithéliales des voies respiratoires. Notre étude révèle que les NPL interagissent fortement avec les muqueuses et délivrent d’une manière accrue les antigènes dans les cellules. Nous avons également montré l'absence de transcytose et de passage paracellulaire des NPL ou des antigènes délivrés dans un modèle de barrière épithéliale in vitro. Les résultats in vivo confirment l'absence de passage nez-cerveau des NPL et montrent qu’elles prolongent fortement le temps de résidence nasale des antigènes qui sont ensuite éliminés par le tractus gastro-intestinal.Ces résultats mettent en évidence l'intérêt des NPL comme vecteurs pour la prochaine génération de médicaments et de vaccins. / Nanoparticles technology for mucosal delivery of vaccines received a growing interest in the last decades. Intranasal administration owns great advantages for immune system stimulation, such as local and systemic protection against infectious diseases. However delivery systems and adjuvants are often required to efficiently trigger mucosal and systemic immune responses. In this thesis, nanoparticles (NP) have been evaluated as delivery system for a nasal universal influenza vaccine in a People Program of the European Union Seventh Framework Program FP7 called UniVacFlu. The aim of the UniVacFlu network is to develop a universal influenza vaccine administered through the mucosal route. We used porous maltodextrin nanoparticles with a lipidic core (NPL). We loaded an adjuvanted antigen named CTA1-3M2e-DD in the NPL. CTA1-3M2e-DD is composed of the A1 subunit of the cholera toxin and a conserved epitope of influenza A virus (M2e), while DD, dimer of the synthetic analogue of the Staphyloccous aureus protein A, targets B cells. Interestingly the antigen loading in NPL was quantitative for the antigen: NPL 1:5 mass ratio and the formulation was stable for at least six months at 4°C. We assessed the successful delivery of the antigen by NPL in airway epithelial cells and macrophages. These formulations are currently evaluated by the UniVacFlu consortium in mice.One of the main issues of intranasal vaccines is the toxicity that can be elicited by the nose-brain passage of one of their components. We investigated the loading of antigens in NPL and their delivery in airway mucosa. We observed a high endocytosis of NPL and an increased protein delivery into the cells. On a transwell model of the airway mucosa we assessed the absence of transcytosis and paracellular passage of the NPL. In vivo results confirmed the lack of nose-brain passage of the NPL, as NPL were found not to cross the mucosa. Interestingly, we observed an increased nasal residence time of the protein targeted by NPL. The particles after having delivered their payload are totally eliminated through the gastrointestinal tract, making these nanoparticles good candidates for mucosal delivery system. These results highlight the interest of NPL as vectors for mucosal delivery of drugs.

A magnetic target drug delivery system consisting of biodegradable polymeric microspheres (poly D, L-lactic acid) loaded with magnetite nanoparticles (10-100 nm) and anticancer drug (paclitaxel) was studied. The magnetite nanoparticles were synthesized by chemical precipitation. The as-synthesized magnetite nanoparticles were subsequently introduced into a mixture of polymer magnetic polymeric composite particles were investigated and further correlated with the reaction parameters. It was found that the size and characteristics of the polymeric composite particles depended on the viscosity of the polymer solution. Preliminary drug release experiments showed that the loaded drug was released with the degradation of the polymer. The release rates could be enhanced by an oscillating external magnetic field. / Singapore-MIT Alliance (SMA)

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

RNA interference (RNAi) is a conservative post-transcriptional gene silencing mechanism that can be mediated by small interfering RNAs (siRNAs). Given the effectiveness and specificity of RNAi, the administration of siRNA molecules is a promising approach to cure diseases caused by abnormal gene expression. However, as siRNA is susceptible to degradation by nucleases and it can hardly penetrate cell membranes due to its polyanionic nature, a successful translation of the RNAi mechanism for therapeutic purposes is contingent on the development of safe and efficient delivery systems. This dissertation described the development of novel siRNA delivery systems on the basis of polymeric and dendrimeric materials and also demonstrated the application of one optimized delivery system to deliver therapeutic siRNAs in a cardiovascular disease model in vivo. We studied a linear peptide polymer made from cell penetrating peptide monomers and investigated the contribution of the polymeric structure, degradability, and ligand conjugation to the siRNA loading capacity, biocompatibility, and transfection efficiency of polymeric materials. With the obtained knowledge and experience, we invented a neutral crosslinked delivery system aiming to solve the inherent drawbacks of traditional cationic delivery systems that are based on electrostatic interactions. The new concept utilized buffering amines to temporarily bind siRNA and a crosslinking reaction to immobilize the formed particles, and targeting ligands modified on the neutral dendrimer surface further enhanced the interactions between the delivery vehicles and target cells. The obtained delivery system allowed stability, safety, controllability, and targeting ability for siRNA delivery, and the method developed here could be transformed to other polymeric or dendrimeric cationic materials to make them safer and more efficient. To exploit the therapeutic potential of siRNA delivery, we developed a tadpole-shaped dendrimeric material to deliver siRNA against an Angiotensin II receptor in a rat ischemia-reperfusion model. Our results showed that the nonaarginine-conjugated tadpole dendrimer was capable of delivering siRNA effectively to cardiac cells both in vitro and in vivo, and the successful down-regulation of the Angiotensin II receptor preserved the cardiac functions and reduced the infarct size post-myocardial infarction. This dissertation paves a way for transforming multifunctional non-viral siRNA delivery systems into potent therapeutic strategies for the management of cardiovascular diseases.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide, accounting for over 600,000 deaths per year. The most common treatment strategy for intermediate and advanced stage unresectable HCC is transarterial chemoembolization (TACE), which involves the local administration of a chemotherapeutic drug combined with arterial occlusion resulting in ischemic tumor necrosis. However, TACE suffers from inadvertent exposure of noncancerous liver parenchyma to embolic agents resulting in liver injury. In some cases, over-embolization has lead to infection, necrosis of unaffected liver tissue, and even liver failure which suggests the need for a biocompatible, multifunctional embolic material which can deliver anticancer drugs with high target specificity. Our laboratory has recently developed a method to fabricate porous silicon (pSi) microparticles with defined physicochemical properties based on photolithography and anodic etching. These microparticles function as multistage drug delivery systems that can circumvent the biobarriers present in the systemic circulation enabling site-specific localization and release of chemotherapy and imaging agents. The versatility of the fabrication process enables the realization of microparticles ranging in size from 600nm to 116[mu]m in diameter with varying shapes, including discoidal, cylindrical and hemispherical, and varying porosity with pore sizes ranging from 6nm to greater than 50nm in diameter. Nanoparticles, such as quantum dots, siRNA-loaded nanoliposomes, gadolinium-based contrast agents, gold and iron oxide nanoparticles, are loaded in pSi microparticles by tailoring their pore sizes and surface chemistries. This thesis presents preliminary results on the applicability of biocompatible, engineered pSi microparticles as an embolic agent for HCC chemoembolization therapy. Hemispherical microparticles with 116[mu]m diameter were successfully fabricated and suspended in phosphate buffered saline (PBS). A microvascular construct was rapid prototyped in polydimethylsiloxane (PDMS) as an in vitro experimental platform to study the embolization behavior of pSi microparticles. Oxidized pSi microparticles were introduced into the microfluidic device at an appropriate flow rate and time-lapse images were taken showing the formation of occlusions at the bifurcation within minutes of administration. Furthermore, penetration through the bifurcation was completely hindered suggesting that pSi microparticles can potentially be used as a biocompatible, multifunctional chemoembolization agent. Although these results are promising, further investigations are warranted.

The aim of this study was to investigate in vitro transdermal diffusion of a small peptide namely arginine vasopressin (AVP) with the aid of the novel PheroidTM drug delivery system. Generally, peptides seem unfit for transdermal permeation, but it was thought prudent to explore the suitability of this lipid-based system after success was achieved with entrapment of tuberculostatics, bacteria and viruses. Bestatin (a selective aminopeptidase inhibitor) was employed to circumvent any skin-related degradation of the active. Therefore, the effect of bestatin on the preservation of AVP during diffusion was investigated. Vertical Franz cell diffusion studies were conducted with female abdominal skin, with AVP at a concentration of 150 pglml in the donor phase and Hepes buffer as the receptor phase over a twelve-hour period. To prove entrapment of AVP within the lipid structures of the PheroidsTM, fluorescentlylabelled samples were monitored by means of confocal laser scanning microscopy (CLSM), which revealed definite entrapment. In vitro permeation profiles for AVP exhibited a biphasic character, with the majority of permeation occurring during the first two hours. The PheroidTM delivery system proved to be advantageous when applied as delivery medium. The inclusion of bestatin has an enhancing effect on permeation probably due to its protection of AVP. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.