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

The aim of the present study was to design different dosage forms as carrier systems to deliver sorafenib to the lung of BXB-23 transgenic mice using different routes of administration. Three dosage forms were used one of them was an oil-in-water emulsion and the oral route was chosen for this experiment. The other delivery system was a liposome preparation for intratracheal instillation. In this case the oral route was considered as a control experiment. The last dosage form was PLGA microspheres. Before sorafenib administration it was important to develop a HPLC method to assess sorafenib absorption after its administration and to determine its concentrations in mouse serum. The HPLC method allowed sorafenib quantification in small volumes (30 µl) of mouse serum and tissues. The developed HPLC method was validated resulting in satisfactory selectivity, good linearity, good accuracy and precision over the concentration range examined. Sorafenib was successfully incorporated in a fat emulsion (o/w) using a traditional method resulting in a white homogenous emulsion and no particle aggregation was observed. Sorafenib exhibited antitumor activity on the lung adenoma in BXB-23 transgenic mice when administered orally (2 mg sorafenib per mouse) in the emulsion preparation. The determined effect was an approximately 29 % reduction in the tumor area of the adenoma foci and a proliferation reduction. In order to improve the pharmacological effects of sorafenib on the lung adenoma in BXB-23 mice, the targeting of sorafenib directly to the site of action (the lung) was an attractive concept. For this purpose the intratracheal route was used. Since sorafenib administration by instillation required incorporation of sorafenib in a dosage form suitable for its lipophilic nature, a liposome suspension was the second dosage form used. A lyophilization method was employed for sorafenib liposome preparation utilizing dilauroylphosphatidylcholine (DLPC) which is safe and tolerable for the lung. Incorporation of sorafenib in the liposomes did not influence the particle size and its distribution. The sorafenib liposomes showed high encapsulation efficiency, good stability at 4 °C for one month and satisfactory in vitro release properties and inhibited Raf-1 mediated activation of ERK in cell culture assay. In a pharmacokinetic experiment sorafenib loaded liposomes were instilled directly into the lung. The results revealed that a significant level of sorafenib was achieved in the lung tissues after 2 hours and then reduced after 48 h and remained nearly constant for one week. On the other hand, only traces of sorafenib were found in the mice serum up to 48 h. Subsequently, the pharmacological activity of sorafenib (1 mg per mouse) was studied when delivered in a liposomal suspension intratracheally to treat the lung adenoma of BXB-23 mice. The data of this experiment demonstrated that sorafenib intratracheal instillation resulted in a reduction of tumor area of adenoma foci (67 %) and an elevation of the percent of apoptotic cells. In contrast, prolongation of the treatment period did not further enhance sorafenib activity on the lung adenoma. This previous finding suggested a development of multidrug resistance (MDR) by the adenoma foci cells against sorafenib instillation, which was examined by immunohistochemistry staining. The percent of MDR positive cells was higher after two and three weeks sorafenib liposome instillation treatment than that after one week treatment. The last dosage form used for sorafenib was microspheres, which were prepared by emulsion-diffusion-evaporation method using biodegradable PLGA 50:50 resulting in a white lyophilized powder. The system was characterized physicochemically and revealed a good microspheres yield, high encapsulation efficiency, a homogenous particle size distribution and slow in vitro release of sorafenib. The other strategy studied in the present research project was gene delivery to target the lung bearing tumor of BXB-23 mice using a non-viral vector (polyethylenimine). Polyethylenimine (PEI) was used to investigate its efficiency in transfecting lung bearing tumor of BXB-23 mice model and its ability to transfect the adenoma foci cells. LacZ, which encodes Beta-galactosidase was used in the present study as a reporter gene and was complexed with PEI before delivered intravenously. A high LacZ expression in the alveolar region with some expression in the adenoma foci was observed. On contrary, a low LacZ expression in the alveoli and in the adenoma foci was achieved after instillation of the same polyplex intratracheally. / Das Ziel der vorliegenden Dissertation war es, verschiedene galenische Darreichungsformen als Trägersystem für Sorafenib zu entwickeln, um den direkten Transport des Arzneistoffes zum Zielorgan Lunge von BXB-23 transgenen Mäusen zu ermöglichen. Für die verschiedenen Applikationswege wurden drei Darreichungsformen gewählt. Eine Öl-in-Wasser-Emulsion sollte oral verabreicht werden. Für die intratracheale Instillation wurde ein liposomales Präparat gewählt. Die letzte Darreichungsform stellten PLGA Mikrosphären dar. Um die Absorption von Sorafenib nach Administration bestimmen zu können, wurde die Konzentration des Arzneistoffes im Mäuseserum gemessen. Zur Quantifizierung von Sorafenib in einem geringen Volumen Serum und in Gewebe wurde eine HPLC-Methode entwickelt und validiert. Sorafenib wurde erfolgreich in eine Fettemulsion (o/w) mittels einer traditionellen Methode eingearbeitet. Nach oraler Verabreichung der Emulsion (2 mg/Maus) zeigte Sorafenib auf Lungenadenome eine Antitumor-Aktivität, wobei eine Reduktion der Tumorfläche der Adenomfoci um etwa 29 % und eine Reduktion der Proliferation verzeichnet werden konnte. Zur Verbesserung der pharmakologischen Effekte von Sorafenib auf die Lungenadenome in BXB-23 Mäusen zu verbessern, sollte Sorafenib direkt dem Zielorgan Lunge zugeführt werden. Zu diesem Zweck wurde der intratracheale Administrationsweg gewählt. Da die Instillation von Sorafenibaufgrund seiner lipophilen Natur nur durch Einschluß in eine andere Darreichungsform zu erreichen ist, wurde für die zweite Darreichungsform eine Liposomen-Suspension verwendet. Für die Zubereitung von Sorafenib in Liposomen wurde eine Lyophilisierungsmethode unter Verwendung von DPLC erarbeitet. Die Einschluss-Effektivität der Sorafenib-beladenen Liposomen war hoch und zeigte bei 4°C eine gute Stabilität für einen Monat. Die erzielten Effekte bei der in vitro Freisetzung und die Hemmung der von Raf1-induzierten Aktivierung von ERK in Zellkulturexperimenten lieferten zufrieden stellende Ergebnisse. In einem pharmakokinetischen Experiment wurden mit Sorafenib beladenen Liposomen direkt in die Lunge appliziert. Die Ergebnisse zeigten, dass nach 2 h eine signifikante Konzentration von Sorafenib im Lungengewebe erreicht wurde. Nach 48 h nahm diese Konzentration ab und blieb dann für eine Woche fast konstant. Andererseits wurden bis zu 48 h nach Gabe des Arzneistoffes nur Spuren von Sorafenib im Mäuseserum gefunden. Folglich wurde die pharmakologische Aktivität von Sorafenib (1 mg/Maus) bei intratrachealer Verabreichung in einer liposomalen Suspension untersucht. Die Ergebnisse zeigten, dass die intratracheale Gabe von Sorafenib eine Reduktion der Tumorfläche der Adenomfoci um 67 % bewirkte, sowie eine Erhöhung des prozentualen Anteils apoptotischer Zellen. Eine Verlängerung der Behandlungszeit zeigte keine zusätzliche Verbesserung der Effekte. Dies lies vermuten, dass hier eine Entwicklung von Multidrug-Resistenz in den Adenomfocizellen gegenüber der Instillation von Sorafenib erfolgte. Dies wurde in immunochemischen Anfärbe-Experimenten untersucht. Die Prozentzahl von MDR-positiven Zellen war nach zwei und drei Wochen Instillation von Sorafenib-Liposomen höher als nach einer Woche. Die letzte verwendete Darreichungsform für Sorafenib waren Mikrosphären, die durch Emulsions-Diffusions-Evaporations-Methoden in biologisch abbaubarem PLGA 50:50 hergestellt wurden. Dies ergab ein weißes, lyophilisiertes Pulver. Das System wurde physiochemisch charakterisiert und ergab ein gutes Mikrosphären-Ergebnis, hohe Einschluss-Effektivität, eine homogene Verteilung der Partikelgrößen und eine langsame in vitro Freisetzung von Sorafenib. Die andere untersuchte Strategie war Gen-Delivery, um den Lungentumor von BXB-23 Mäusen mittels eines nicht-viralen Vektors (Polyethylenimin, PEI) anzuzielen. PEI wurde verwendet, um die Effektivität der Transfektion des Lungentumors zu untersuchen und seine Fähigkeit, die Adenomfocizellen zu transfizieren. LacZ, das Beta-Galactosidase codiert, diente bei diesem Experiment als Reportergen und wurde vor intravenöser Gabe mit PEI komplexiert. Eine hohe LacZ-Expression in der alveolaren Region, aber nur eine geringe Expression in den Adenomfoci wurde beobachtet. Im Gegensatz dazu wurde eine geringe Expression von LacZ in den Alveolen und den Adenomfoci nach intratrachealer Instillation des gleichen Polyplex erreicht.

Thesis advisor: Cyril P. Opeil / The physiology of the vasculature in the central nervous system (CNS) which includes the blood-brain-barrier (BBB) and other factors, prevents the transport of most anticancer agents to the brain and restricts delivery to infiltrating brain tumors. The heterogeneous vascular permeability in tumor vessels (blood-tumor barrier; BTB), along with several other factors, creates additional hurdles for drug treatment of brain tumors. Different methods have been used to bypass the BBB/BTB, but they have their own limitations such as being invasive, non-targeted or requiring the formulation of new drugs. Magnetic Resonance Imaging guided Focused Ultrasound (MRIgFUS), when combined with circulating microbubbles, is an emerging noninvasive method to temporarily permeabilize the BBB and BTB. The purpose of this thesis was to use this alternative approach to deliver chemotherapeutic agents through the BBB/BTB for brain tumor treatment in a rodent model to overcome the hinderances encountered in prior approaches tested for drug delivery in the CNS. The results presented in thesis demonstrate that MRIgFUS can be used to achieve consistent and reproducible BBB/BTB disruption in rats. It enabled us to achieve clinically-relevant concentrations of doxorubicin (~ 4.8±0.5 µg/g) delivered to the brain with the sonication parameters (0.69 MHz; 0.55 MPa; 10 ms bursts; 1 Hz PRF; 60 s duration), microbubble concentration (Definity, 10 µl/kg), and liposomoal doxorubicin (Lipo-DOX) dose (5.67 mg/kg) used. The resulting doxorubicin concentration was reduced by 32% when the agent was injected 10 minute after the last sonication. Three weekly sessions of FUS and Lipo-DOX appeared to be safe in the rat brain, despite some minor tissue damage. Importantly, the severe neurotoxicity seen in earlier works using other approaches does not appear to occur with delivery via FUS-BBB disruption. The resuls from three weekly treatments of FUS and Lipo-DOX in a rat glioma model are highly promising since they demonstrated that the method significantly inhibits tumor growth and improves survival. Animals that received three weekly sessions of FUS + Lipo-DOX (N = 8) had a median survival time that was increased significantly (P<0.001) compared to animals who received Lipo-DOX only (N = 6), FUS only (N = 8), or no treatment (N = 7). Median survival for animals that received FUS + Lipo-DOX was increased by 100% relative to untreated controls, whereas animals who received Lipo-DOX alone had only a 16% improvement. Animals who received only FUS showed no improvement. No tumor cells were found in histology in 4/8 animals in the FUS + Lipo-DOX group, and only a few tumor cells were detected in two animals. Tumor doxorubicin concentrations increased monotonically (823±600, 1817±732 and 2432±448 ng/g) in the control tumors at 9, 14 and 17 days respectively after administration of Lipo-DOX. With FUS-induced BTB disruption, the doxorubicin concentrations were enhanced significantly (P<0.05, P<0.01, and P<0.0001 at days 9, 14, and 17, respectively) and were greater than the control tumors by a factor of two or more (2222±784, 3687±796 and 5658±821 ng/g) regardless of the stage of tumor growth. The transfer coefficient Ktrans was significantly (p<0.05) enhanced compared to control tumors only at day 9 but not at day 14 or 17. These results suggest that FUS-induced enhancements in tumor drug delivery for Lipo-DOX are relatively consistent over time, at least in this tumor model. These results are encouraging for the use of large drug carriers, as they suggest that even large/late-stage tumors can benefit from FUS-induced drug enhancement. Corresponding enhancements in Ktrans were found variable in large/late-stage tumors and not significantly different than controls, perhaps reflecting the size mismatch between the liposomal drug (~100 nm) and Gd-DTPA (molecular weight: 938 Da). Overall, this thesis research provides pre-clinical data toward the development of MRIgFUS as a noninvasive method for the delivery of agents such as Lipo-DOX across the BBB/BTB to treat patients with diseases of the central nervous system. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Physics.

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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.

O perfil de liberação e o efeito de um sistema de liberação prolongada contendo metronidazol, antimicrobiano prescrevido para o tratamento da periodontite, foram avaliados na presença de biofilmes supra e subgengivais, representados respectivamente pelas bactérias Streptococcus mutans e Porphyromonas gingivalis. Os biofilmes foram crescidos e expostos ao sistema de liberação prolongada contendo metronidazol (MDZ) ou ao controle de veículo da formulação (CV), composto de monoglicerídeos e monoesterato de sorbitano. Biofilmes não tratados foram utilizados como controle negativo (CN). Os biofilmes e os meios de cultura de S. mutans foram coletados após a primeira exposição aos tratamentos nos tempos 24, 48, 72 e 96 horas enquanto para biofilmes de P. gingivalis os tempos foram 24, 48 e 72 horas. Após coleta, os biofilmes foram analisados em relação à quantificação de fármaco e viabilidade bacteriana (biofilmes de S. mutans: n=3; biofilmes de P. gingivalis: n=6). Biofilmes de S. mutans também foram avaliados em relação à acidogenicidade. Nos biofilmes supragengivais, a quantificação de MDZ nas primeiras 24 horas foi de 7% em relação à concentração inicial de fármaco na formulação, permanecendo em torno de 1% para os demais tempos. O teor de MDZ liberado da formulação reduziu a viabilidade bacteriana no tempo 24 horas e diminuiu a acidogenicidade dos biofilmes por 48 horas em relação aos grupos CV e NC (p<0,05). Já para os biofilmes subgengivais, 19% de MDZ foram liberados da formulação nas primeiras 24 horas e 5% do fármaco foram quantificados nas análises dos demais tempos. O antimicrobiano liberado reduziu a viabilidade bacteriana em todos os tempos em relação à CV e NC (p<0,05), não sendo diferente estatisticamente entre si (p>0,05). O grupo CV apresentou menor viabilidade bacteriana se comparado ao grupo CN (p<0,05), mas maior viabilidade em comparação ao grupo MDZ em todos os tempos (p<0,05). De uma forma geral, o sistema de liberação prolongada proposto neste estudo foi capaz de inviabilizar a proliferação de biofilmes de P. gingivalis e de desestabilizar biofilmes de S. mutans. Além disso, os microambientes originados pelos biofilmes interferiram na cinética de liberação do metronidazol, diminuindo sua disponibilidade biológica. Assim, considerando a continuidade do biofilme sub e supragengival, torna-se interessante aprofundar os estudos sobre formulações que possam inibir biofilmes subgengivais ao mesmo tempo em que desestabilizam biofilmes supragengivais, evitando a rápida recolonização dos nichos periodontais tratados. Em acréscimo, a possibilidade de estudar parâmetros operacionais de desenvolvimento da formulação farmacêutica utilizando-se modelos de biofilmes patogênicos pode ser considerada em futuros estudos / The drug release profile and the effect of a controlled release system containing metronidazole, an antibiotic prescribed for the treatment of periodontitis, were evaluated in the presence of supra and subgingival biofilms, represented respectively by the bacteria - Streptococcus mutans and Porphyromonas gingivalis. The biofilms were grown and exposed to the controlled release system containing metronidazole (MDZ) as well as vehicle control (VC) of the formulation containing monoglycerides and sorbitan monostearate. Untreated biofilms were used as negative control (NC). The biofilms and culture media of S. mutans were collected after the first exposure to the treatments at times 24, 48, 72 and 96 hours while for P. gingivalis biofilms, the times were 24, 48 and 72 hours. After collection, biofilms were analyzed for drug quantification and bacterial viability (S. mutans biofilms: n=3; P. gingivalis biofilms: n=6). Biofilms of S. mutans were also evaluated for acidogenicity. In the supragingival biofilms, the MDZ quantification in the first 24 hours was 7% in relation to the initial concentration of drug in the formulation, remaining around 1% for the remaining times. The amount of MDZ released from the formulation reduced the bacterial viability in the 24 hour and decreased the acidogenicity of the biofilms by 48 hours in relation to the VC and NC groups (p <0.05). As for subgingival biofilms, 19% of MDZ was released from the formulation in the first 24 hours and 5% of the drug was quantified in the other analyses. The MDZ released reduced bacterial viability in relation to VC and NC (p <0.05), and was not statistically different at all sampling times studied (p> 0.05). The VC group presented lower number of viable bacteria than NC (p <0.05), however, it was higher when compared to the MDZ-treated group at all sampling times studied (p <0.05). In general, the controlled release system proposed in this study was able to prevent the proliferation of P. gingivalis biofilms and to destabilize S. mutans biofilms. In addition, microenvironments caused by biofilms interfered with the release kinetics of metronidazole, reducing its bioavailability. Thus, considering the continuity of the sub and supragingival biofilms, it is imperative to deepen the studies on formulations that can inhibit the formation of subgingival biofilms while destabilizing supragingival biofilms, thereby avoiding the rapid recolonization of the treated periodontal niches. In addition, the possibility of studying operational parameters for the development of pharmaceutical formulations using models of pathogenic biofilms can be considered in future studies

With recent development of nanoscience and nanotechnology, a great amount of efforts have been devoted to nanomedicine development. Among various nanomaterials, silica nanoparticle (NP) is generally accepted as non-toxic, and can provide a versatile platform for drug loading. In addition, the surface of the silica NP is hydrophilic, being favorable for cellular uptake. Therefore, it is considered as one of the most promising candidates to serve as carriers for drugs. / The present thesis mainly focuses on the design of silica based nanocarrier-drug systems, aiming at achieving safe nanocarrier excretion from the biological system and enhanced drug efficacy, which two are considered as most important issues in nanomedicine development. / To address the safe carrier excretion issue, we have developed a special type of self-decomposable SiO₂-drug composite NPs. By creating a radial concentration gradient of drug in the NP, the drug release occurred simultaneously with the silica carrier decomposition. Such unique characteristic was different from the conventional dense SiO₂-drug NP, in which drug was uniformly distributed and can hardly escape the carrier. We found that the controllable release of the drug was primarily determined by diffusion, which was caused by the radial drug concentration gradient in the NP. Escape of the drug molecules then triggered the silica carrier decomposition, which started from the center of the NP and eventually led to its complete fragmentation. The small size of the final carrier fragments enabled their easy excretion via renal systems. / Apart from the feature of safe carrier excretion, we also found the controlled release of drugs contribute significantly to the drug efficacy enhancement. By loading an anticancer drug doxorubicin (Dox) to the decomposable SiO₂-methylene blue (MB) NPs, we achieved a self-decomposable SiO₂(MB)-Dox nanomedicine. The gradual escape of drug molecules from NPs and their enabled cytosolic release by optical switch, led to not only high but also stable drug concentration in cytosol within a sustained period. This resulted in enhanced drug efficacy, which is especially manifested in multidrug resistant (MDR) cancer cells, due to the fact that the NP-carrier drug can efficiently bypass the efflux mechanisms and increase drug availability. Together with its feature of spontaneous carrier decomposition and safe excretion, this type of nanomedicine’s high drug efficacy highlights its potential for low dose anticancer drug treatment and reduced adverse effect to biological system, holding great promise for clinical translation. / The enhanced drug efficacy by employing the self-decomposable silica nanocarrier is also demonstrated in photodynamic therapy (PDT). The loose and fragmentable features of the self-decomposable SiO₂-photosensitizer (PS) NPs promoted the out-diffusion of the generated ROS, which resulted in a higher efficacy than that of dense SiO₂-PS NPs. On the other hand, we also explored another nanocarrier configuration of Au nanorods decorated SiO₂ NP, with PS drug embedded into dense SiO₂ matrix. A different mechanism of drug efficacy enhancement was presented as the Au’s surface plasmon resonance enhanced the ROS production. Although the drug efficacy of such SiO₂(PS)-Au NPs was similar to that of self-decomposable SiO₂-PS NPs, their potential for clinical applications was limited without the feature of safe carrier excretion. / In summary, the self-decomposable SiO₂ based NP developed is a most promising system to serve as safe and effective carriers for drugs. Together with the known biocompatibility of silica, the feature of controllable drug release and simultaneous carrier decomposition achieved in the self-decomposable SiO₂-drug NPs make it ideal for a wide range of therapeutic applications. / 隨著近年來納米科學技術的快速發展，致力於納米藥物的研发也越來越多。在眾多納米材料體系中，二氧化硅納米顆粒因其無毒、易載藥、且易於細胞攝入等特性，被認為是最具前景的藥物載體之一。 / 本文主要致力於設計以二氧化硅納米顆粒為載體的納米藥物體系，使之同時具備能夠被生物體安全排泄以降低潛在不良影響，并且能夠加強藥效的特性，而這兩方面被認為正是納米藥物發展中最重要的議題。 / 爲了實現藥物載體安全排泄，我們設計了一種特殊類型可自降解的二氧化硅-藥物複合納米顆粒。通過在納米顆粒中控制形成徑向藥物濃度梯度分佈，我們達到了藥物釋放的同時伴隨二氧化硅載體解體的效果。這一特徵不同於傳統二氧化硅-藥物複合納米顆粒中藥物均勻分佈而難以擴散出載體的情況。我們發現在這種可自降解的二氧化硅-藥物複合納米顆粒中，首先徑向藥物濃度梯度分佈所引起的擴散控制著藥物釋放，而後藥物分子的流失促發二氧化硅載體由內而外的逐步分解，最終全面解體分裂成碎片。這些碎片的小尺度使得它們易於經泌尿系統安全排泄出體外。 / 除此之外，我們發現這種納米載體的可控藥物釋放特性可以大大提高藥效。通過將抗癌藥阿黴素載入自降解二氧化硅-亞甲藍納米顆粒中，我們得到一種可自降解二氧化硅/亞甲藍-阿黴素(SiO₂(MB)-Dox)複合納米顆粒。藥物分子可以逐漸擴散出納米顆粒，並且在光控開關作用下釋放到細胞胞漿中，使之在胞漿中持續保持穩定高濃度。這樣使得藥效得以加強，尤其是在多藥抗藥性腫瘤細胞中作用尤為明顯，這得益於納米載體藥物可以有效避開藥泵機制并提高藥物利用率。除了它的自發載體分解和安全排泄特性，這種納米藥物的高藥效使得它在低藥量治療和減少不良副作用方面的潛力突出，臨床應用前景廣大。 / 可自降解二氧化硅納米載體所帶來的的藥效增強亦顯示在光動力學治療法中。可自降解二氧化硅-光敏劑藥物(SiO₂-PS)複合納米顆粒鬆散易分解的結構特性促使其內部產生的活性氧物質易於擴散出藥物載體，這使得它的藥效高於傳統二氧化硅-光敏劑複合納米顆粒。另一方便，我們設計了一種金修飾的二氧化硅納米顆粒載體。它具有另一種不同的藥效增強機制，即利用金納米顆粒表面等離子體共振效應來增強活性氧物質的產生。雖然藥效與可自降解二氧化硅-光敏劑複合納米顆粒相似，但是它無法安全排泄，限制了其在臨床上的應用。 / 綜上所述，我們發展的可自降解二氧化硅納米顆粒作為一種安全高效的藥物載體顯示出其非常大的應用前景。二氧化硅以其衆所周知的生物相容性，和我們發展的可控藥物釋放及同步載體分解特性，已成為理想的藥物載體并有希望廣泛適用於治療應用。 / Zhang, Silu = 基於無定二氧化硅納米顆粒的安全高效納米藥物的研究 / 張思鷺. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references. / Abstracts also in Chinese. / Title from PDF title page (viewed on 12, October, 2016). / Zhang, Silu = Ji yu wu ding er yang hua gui na mi ke li de an quan gao xiao na mi yao wu de yan jiu / Zhang Silu. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Organosilica nanoparticles have attracted a lot of research interest in a variety of areas such as drug delivery and catalysis because of their properties which include high surface area as well as tunable particle and pore size. In particular, nanoparticles with large pore sizes are of great interest because of their potential to host large guest molecules such as proteins and as catalysts. The focus of the work in the thesis was to develop phosphonate functionalized organosilica nanoparticles for biomedical and catalytic applications. Raspberry textured phosphonatesilica nanoparticles denoted, RNPPME(2.5) (where the number in the brackets represents the moles of organophosphonate per gram), with large pore size (11–17 nm), uniform particle size (70 – 90 nm) and high surface area were produced through the use of template directed base catalysed synthesis, using tetraethylorthosilicate (TEOS) and dimethylphosphonatoethyltrimethoxysilane (DMPTMS) as the silica sources. The role of the reaction conditions such as temperature, surfactant concentration, pH, organosilane concentration and type were investigated and a mechanism for the raspberry nanoparticle formation was proposed. The particles were characterized using electron microscopy (SEM and TEM), Dynamic light scattering (DLS), silicon and phosphorus solid state NMR, and solution phase proton NMR of base digested particles, FT–IR, nitrogen adsorption porosimetry and thermal analysis (TGA). The ability of the particles to host protein molecules of the model protein, bovine serum albumin (BSA) was investigated and the particle–protein composite was characterized using circular dichroism (CD). Raspberry textured nanoparticles were found to host large quantities (26 wt%) of protein. Studies on other (small pore (3 nm) phosphonate functionalized nanoparticles NP_PME(0.2) and NP_PME(1.0)) and (3 nm pore) unfunctionalized mesoporous silica nanoparticles (MSN) revealed that phosphonate loading and the pore size influenced the protein uptake In addition to high protein uptake, the RNP_PME(2.5) particles also absorbed protein molecules rapidly (~ 20 minutes to maximum load). CD studies determined that the particle bound protein structure was not affected at physiological pH (7.40). The vast majority of the previously reported studies involving protein immobilization involved the use of bulk silica materials, which cannot be dispersed and hence those materials were unsuitable for in vivo protein delivery applications. The BSA@RNP_PME(2.5) particles showed good protein load and dispersion properties and hence are excellent protein delivery agents. Dispersions of nanoparticle composite BSA#@RNP*_PME(2.5) (where BSA# represents fluorescein isothiocyanate labelled BSA and RNP*_PME(2.5) represents rhodamine B isothiocyanate labelled RNP_PME(2.5)) was used to successfully deliver membrane impermeable protein BSA into HeLa cells. Intracellular protein delivery has attracted great interest due to its potential therapeutic applications and research tool value (e.g. for studying various cellular pathways). The toxicity of the guest free particles RNP*_PME(2.5) and the protein loaded particles BSA#@RNP*_PME(2.5) was studied using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The particles and the protein-particle composite were found to be non-toxic. The mechanism of the particle uptake by the cells was also studied. The unloaded (protein free) particles were found to be taken up by caveolar endocytosis pathway and the protein loaded particles were taken up by folic acid mediated pathway. The results indicated that the particles can successfully deliver membrane impermeable protein across the cell membrane. This result suggested that the particles can potentially be used for intracellular protein delivery applications. Raspberry textured nanoparticles RNP_PME(2.5) were also used to host the enzyme lipase. It was demonstrated that immobilization increased the maximum velocity and Michaelis constant of the enzyme and also that the particles offered protection against the denaturing agent, urea. Finally, in a chemical catalysis application, the RNP_PME(2.5) particles were used to synthesize the platform chemical HMF, through Brönsted acid catalysed dehydration of fructose. High yields of HMF (87 %) were achieved when 10 wt% fructose was used. The particles demonstrated good recyclability and also the ability to convert up to 50 wt% fructose into HMF (80 % yield). The particles therefore acted as protective agents for enzymes and can therefore be used as enzyme immobilizing agents. Additionally, they also acted as excellent Brönsted acid catalysts.

The field of nanomedicine has witnessed an incredible expansion, from a total market value in 2003 of $500 million expected to rise to $160 billion by 2015 (Global Industry Analysts, Inc.). The nanomedicine industry is forecasted to grow and have a significant impact on the economy, with sectors such as biomaterials, diagnostics and drug delivery expected to play a major role. This thesis gives a detailed account of the synthesis and characterisation of molecularly imprinted nanogels for drug delivery. Their toxicity and potential use as a targeted carrier to cancerous cells is evaluated. Initially an overview of nanomaterials and their uses in many areas such as agriculture, energy storage and technology are discussed. The impact of nanomaterials on the life sciences is examined; in particular their application in drug delivery is focussed upon. Chapters 2, 3 and 4 make up the results and discussion of this work. Chapter 2 focuses on developing the synthesis of the acrylamide based nanogels and, vitally, incorporating a suitable fluorescent tag in order to track the nanogels in vitro and in vivo. Fundamentally toxicity studies carried out on the nanogels, both in vitro and in vivo in Danio rerio (zebrafish) are reported in Chapter 3 to ensure the nanogels are biocompatible. Chapter 4 introduces an innovative approach, molecular imprinting, to incorporating a drug into the nanogels. The upload and release of Tamoxifen (a drug used to treat breast cancer) at reduced pH, was also analysed. Finally future development of the carrier is discussed and key issues that need to be addressed.

Polyelectrolyte microcapsules were first established in 1998 as a potential drug delivery vehicle. Despite being well-established, microcapsules have not yet been thoroughly considered as a viable means of targeted drug delivery. This is largely due to the fact that microcapsules are inherently prone to unspecific binding to cells and proteins. Targeted delivery of drugs to specific diseased sites in the body is an area of research that has attracted many studies, particularly in drug deliveries that utilise microparticles. By achieving targeted delivery of a drug, one can increase the efficacy of the treatment, thus, reducing unwanted side effects. This thesis investigates methods which can modify these microcapsules in order to fine tune the release of the encapsulated drug as well as site-specific delivery of these vesicles i.e. obtain spatiotemporal control. To this end, biodegradable microcapsules of varying constituents are manufactured and their biodegradability is indirectly measured through quantification of the release of an encapsulated fluorescent protein (Rhodamine B-BSA). Fluorometry analysis of the supernatants of these microcapsule suspensions indicated that microcapsules synthesised from poly-L-arginine and poly-L-glutamic acid have the ability to encapsulate bovine serum albumin (BSA) with a high encapsulation efficiency (79.7%). Furthermore, they are able to produce a sustained release of BSA over a period of 5 Days. To complement this controlled-release study, an investigation into self-degradable microcapsules was undertaken. To achieve this, proteinase was encapsulated in both biodegradable and non-biodegradable microcapsules of different thickness. Analysis of the protein release over a period of 24 hours revealed that the release profiles of these microcapsules can be successfully controlled. Biodegradable microcapsules released 87% more protein than their non-biodegradable counterpart after 2 hours of incubation in deionised water. This provides conclusive evidence that the biodegradable microcapsules were, indeed, self-degradable. The latter part of this thesis focuses on achieving specific and exclusive targeted delivery using polyelectrolyte microcapsules, with respect to protein substrates. This is accomplished by creating an antibody-functionalised poly(ethylene glycol) (PEG) assembly within the microcapsule structure. Site-specific adsorption of these microcapsules is tested using protein micropatterns. Results obtained from adsorption assays using anti-collagen type IV-functionalised microcapsules show a 600-fold increase in binding to collagen type IV islands, compared to control proteins (fibronectin and BSA). This proves that significant adsorption was achieved on the target protein, with unspecific adsorptions being heavily suppressed on control proteins. Furthermore, similar results were found when microcapsules were functionalised with anti-fibronectin and exposed to fibronectin, highlighting the versatility of this type of biofunctionalisation.