First part of this thesis was focused on the previously overlooked field of C1'-branched acyclic nucleoside phosphonates (ANPs). Five diverse synthetic approaches were developed/optimized affording key 6-chloropurine intermediates bearing N9 -phosphonomethoxyethyl (PME) branched at C1' position in 2-4 steps. It was demonstrated that these intermediates can be further vastly diversified into ANPs bearing both natural and unnatural nucleobases. Single enantiomers as well as racemates of final C1'-branched ANPs (overall 48 final compounds) were prepared and selected compounds were evaluated with respect to their biological properties. The aforementioned ANPs showed no antiviral potency against studied viruses and only weak to moderate cytostatic activity. Adenine C1'-branched ANPs proved to be the most potent currently known inhibitors of Trypanosoma brucei adenine phosphoribosyl transferase (TbrAPRT), an enzyme involved in purine salvage pathway (PSP) of T. brucei. Further biological evaluation of prepared compounds is in progress. Second part of this thesis was focused on development of novel prodrug moieties with higher selectivity index (i.e. toxicity/potency ratio - SI) based on so-called ProTide prodrugs where phenol (present in ProTides) was replaced by tyrosine derivatives. Tenofovir was...

The water solubility of drug molecules plays an important role in consideration of formulation development to treat a wide range of diseases. In this project, two kinds of drug delivery systems, cyclodextrins and liposomes, were developed for insoluble drug delivery to treat Alzheimer's disease (AD) and colorectal cancer (CRC), respectively. AD is an irreversible neurodegenerative disorder associated with the accumulation of amyloid-beta (A??) fibrils. Approximately 10% of people aged 65 and above have AD. Crocetin (CRT) is an active compound isolated from the fruits of gardenia (Gardenia jasminoides Ellis) and the stigmas of saffron (Crocus sativus L.). It has been reported to show various neuroprotective activities. However, poor water solubility and bioavailability are the major obstacles in developing pharmaceutical formulations of CRT. To address the issues, CRT liposomal formulations and CRT-cyclodextrin inclusion complexes were developed and evaluated. CRT-cyclodextrin inclusion complexes significantly increased the water solubility of CRT from the range ??g/mL to mg/mL. The CRT-??-cyclodextrin inclusion complex (1:3 molar ratio of CRT/??-cyclodextrin) was chosen for further studies as it showed the highest encapsulation efficiency (94.73 ?? 0.86%). The formulation had no toxicity to neuronal cells nor AD model cells within the experimental concentration range (0.625 to 100 ??M of CRT). It could downregulate the expression of C-terminus fragments and decrease both intracellular and extracellular levels of A??, which are hallmarks of AD. It also showed dose-dependent neuroprotective and antioxidant effects against H2O2-induced cell death. Pharmacokinetics and biodistribution studies showed that this CRT-??-cyclodextrin inclusion complex was suitable for intravenous administration. The formulation significantly increased the bioavailability of CRT and facilitated CRT crossing the blood-brain barrier to enter the brain. Similar to AD, CRC is increasingly prevalent with aging populations. Approximately 60% of CRC patients are aged 70 and above. Halofuginone (HF) is an active pharmaceutical ingredient (API) originated from Chinese quinine (Dichroa febrifuga Lour.) and artesunate (ART) is a semi-synthetic derivative of artemisinin (ATS) extracted from annual wormwood (Artemisia annua L.). Both APIs show anticancer activities by inhibiting the growth of CRC. However, low aqueous stability limits their applications. Liposome formulation with surface functionalization by CPP2 cell-penetrating peptide was developed to deliver HF and ART for targeted CRC therapy. CPP2 is a peptide that can selectively penetrate colon cancer cells. The liposomal drug formulations had uniform particle size (about 100 nm), high encapsulation efficiency (over 80%) and good stability upon 14 days of storage. In cellular uptake study, CPP2-modified liposome showed stronger permeability and selectivity to colon cancer lines without inducing lysosomal degradation. CPP2 surface-modified liposomal drugs demonstrated greater anticancer activities than free form of drugs or conventional liposomal drugs. Combinations of HF and ART formulations notably decreased cancer cell viability as compared to single formulation alone, which indicated that HF and ART formulations exhibited synergistic anticancer effects at specific ratios. To conclude, the drug delivery systems, cyclodextrins and peptide-modified liposomes, which were developed for AD and CRC treatment, successfully improved the aqueous solubility of insoluble APIs extracted from Chinese medicinal plants.

La thèse est intitulée « Modifications chimiques de polysaccharides pour l'obtention de nouveaux systèmes polymère-principe actif ». Le thème de la recherche porte sur trois axes principaux, dont l’objectif majeur est la synthèse et la caractérisation de nouveaux systèmes pour la libération contrôlée de principes actifs. Le premier chapitre traite de l’optimisation de la méthode pour préparer des émulsions stables simples inverses (eau/huile) et multiples (eau/huile/eau) à base de polysaccharides (amidon et 2-hydroxyethyle cellulose). On a étudié le processus de relargage de la caféine, l’influence de la présence d’amidon dans la phase aqueuse sur la stabilité et cinétique de libérations. Le second chapitre a pour objet l’obtention de nouveaux principes actifs à partir d’aminoacides et leur immobilisation sur polysaccharides par couplage chimique covalent. Les conjugues polymère-médicament obtenus ont été caractérisés par spectroscopie, physico-chimie. Leur activité antimicrobienne, leur toxicité in vivo, l’efficacité de l’immobilisation et la cinétique de relargage in vitro de médicaments couplés ont été étudiées. Le dernier chapitre traite de la synthèse des dérivés de chitosane avec l’anhydride maléique et l’acide acrylique, ainsi que leur utilisation dans la préparation d’hydrogels sous forme de films et de nanoparticules par copolymérisation avec le méthacrylate de 2-hydroxyethyle et l’acrylamide de N- isopropyl. Les dérivés ont été évalués par leur degré de fonctionnalisation. Les nanosystèmes ont été caractérisés par leur potentiel zêta, leur capacité de gonflement, d’adhésion et de relargage in vitro. Tous les résultats expérimentaux ont été soutenus par importantes études bibliographiques / The thesis is entitled “Chemical modifications of polysaccharides for the obtaining of new drug-polymer systems“. The topic of research includes four main working axes, diverted from the major objective to synthesize and characterize new drug delivery systems. The first chapter treats the optimization of the preparation method of stable simple W/O emulsions and multiple W/O/W emulsions, based on polysaccharides (starch and 2-hydroxyethyle cellulose). We also studied the process of caffeine release outside the multiple emulsions particles and the influence of starch in the internal aqueous phase stage upon the stability and the drug release kinetics. The second chapter studies the obtaining of new active principles from aminoacid and their immobilization on polysaccharides by covalent coupling. The new drug-polymer conjugates were characterized by spectral, physicochemical properties, antimicrobial activity, in vivo toxicity, immobilization efficiency and I vitro drug release kinetics. The last chapter treats the synthesis of the chitosan derivatives with maleic anhydride or acrylic acid, and their use in the preparation of hydrogels as film and nanoparticles by copolymerization with 2-hydroxymethacrilate or N-isopropyl acrylamide. The derivatives were evaluated by determining the degree of functionalization and by spectral methods. The nanosystems were characterized by measurements of zeta potential, swelling and adhesion capacity and membership and in vitro drug release kinetics. All experimental results were supported by important bibliographic studies

Current chemotherapeutics pose many di sadvantages due to their lack of specificity and low therapeutic index. To overcome these challenges, research has focused its attention on the development of nano-based delivery systems that can penetrate the leaky vasculature of tumor endothelium, use site-directed ligands that can bind with high affinity and specific ity to tumor cells, physically entrap poorly soluble drugs, and deliver these cytotoxic agents directly to the tumor site. One approach to nanosystem drug delivery is with the use of peptide amphiphiles (PAs) that are conjugated with the Arginine-Glycine-Aspartic Acid (RGD) motif to actively target a αVβ3 integrin receptors on cancer cells or tumor endothelium. The current work is focused on mechanistic studies to evaluate the uptake of novel RGD amphiphi les with varying alkyl chain lengths (palmitic acid : Cl 6 and stearic acid: C 18) and hydrophilic linkers, 8-amino- 3,6-dioxaoctonoic acid (ADA) or glucose, as micellar delivery systems of hydrophobic anticancer agents. PAs were confirmed for their self-assembling properties and further evaluated for their RGD-mediated binding specificity to purified αVβ3 integrin through a competitive binding fluorescence polarization assay (with novel RGD micelles displacing an integrin-bound fluorescent RGD probe by as much as 63.03%). Ultimately, these nanocarriers were assessed for their ability to deliver phys ically entrapped fluorescein isoth iocyanate (FITC) to A2058 cells overexpressing αVβ3 integrin receptors. Results from confocal microscopy indicate that uptake of RGD micelles was driven by an energy-dependent mechanism, as statistically significant levels of FITC internalization was seen at 37°C versus 4°C (p-value<0.05 for all treatment groups); moreover, intracellular fluorescence was notably higher (as much as 4-fold) when delivered through novel RGD conjugates as opposed to its free form. Regardless of chain length and the number of hydrophilic linkers, all RGD PAs showed promising results as micellar carriers that can effectively deliver their payload to the target tumor site via receptor mediated endocytosis.

No description available.

Transdermal drug delivery systems (TDDSs) play important roles in therapy due to distinct advantages over other forms and types of drug application. While common TDDS patches mainly consist of polymeric matrices so far, inorganic carriers show numerous advantages such as high mechanical stability, possible re-use and re-loading of drugs, and a broad chemical compatibility with therapeutically relevant compounds and chemical enhancers. Mesoporous glasses can be prepared in different monolithic shapes, and offer a particularly wide range of possible pore volumes, pore diameters, and specific surface areas. Further, they show high loading capacities and favorable physical, technical, and biological properties. Here, we explored for the first time monolithic SiO2- based carriers as sustained release systems of therapeutic drugs. In an ideally stirred vessel as model system, we systematically analyzed the influence of pore diameter, pore volume, and the dimensions of glass monoliths on the loading and sustained release of different drugs, including anastrozole, xylazine, imiquimod, levetiracetam, and flunixin. Through multilinear regression, we calculated the influence of different parameters on drug loading and diffusion coefficients. The systematic variation of the mesoporous glass properties revealed pore volumes and drug loading concentrations, but not pore diameter or pore surface area as important parameters of drug loading and release kinetics. Other relevant effectors include the occurrence of lateral diffusion within the carrier and drug-specific properties such as adsorption. The structure–property relationships derived from our data will allow further fine-tuning of the systems according to their desired properties as TDDS, thus guiding towards optimal systems for their use in transdermal drug applications

Yes / Understanding structure-property relationships is critical for the development of new drug delivery systems. This study investigates the properties of Pluronic smart hydrogel formulations for future use as injectable controlled drug carriers. The smart hydrogels promise to enhance patient compliance, decrease side effects and reduce dose and frequency. Pharmaceutically, these systems are attractive due to their unique sol-gel phase transition in the body, biocompatibility, safety and injectability as solutions before transforming into gel matrices at body temperature. We quantify the structural changes of F127 systems under controlled temperature after flow, as experienced during real bodily injection. Empirical formulae combining the coupled thermal and shear dependency are produced to aid future application of these systems. Induced structural transitions measured in-situ by small angle x-ray and neutron scattering reveal mixed oriented structures that can be exploited to tailor the drug release profile.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The combination of natural and synthetic polymers allow for the synthesis of advanced hybrid copolymers. These hybrid copolymers have applications in biomedical areas, one such area being in drug delivery systems (DDS). In this study, a modular approach was used to prepare amphiphilic block copolymers with the ability to self-assemble into three dimensional structures. Reversible addition-fragmentation chain transfer (RAFT) was the synthetic tool used to mediate the polymerization of N-vinylpyrrolidone. RAFT is a versatile method to prepare polymers with control over molecular weight and dispersity. A xanthate chain transfer agent (CTA) was used to obtain the hydrophilic poly(N-vinylpyrrolidone) (PVP) block. An aldehyde functionality could be introduced due to the lability of the xanthate moiety, the procedure of which was effectively optimized to produce quantitative conversion. A dixanthate CTA was synthesized to produce a PVP chain which after the modification reaction, resulted in a α,ω-telechelic polymer. A polypeptide was synthesized via the ring-opening polymerization of Ncarboxyanhydrides (ROP NCA). The living and controllable ROP of NCAs is a method which results in polypeptides, but without a well-defined amino acid order. Poly(γ- benzyl-L-glutamate) (PBLG) was synthesized with a narrow dispersity (Đ = 1.10 – 1.15) using conditions that promote the retention of a terminal primary amine. A protected cysteine functionality was introduced via the terminal amine PBLG chain-end, using peptide synthesis techniques. This resulted in the conjugation of the aldehyde functional PVP and the cysteine terminal PBLG using a covalent, non-reducible thiazolidine linkage. The deprotection of the cysteine, more specifically the deprotection of the thiol was a non-trivial procedure. The thiol protecting acetamidomethyl (Acm) group could not be cleaved using traditional methods, but instead a modified procedure was developed to effectively remove the Acm group while inhibiting hydrolysis of the benzyl esters. It was determined that the conjugation reaction could effectively proceed in N,Ndimethylformamide (DMF) at a slightly elevated temperature and so continued to prepare the amphiphilic hybrid block copolymers, PVP-b-PBLG. A structurally different PBLG chain, namely PBLG-b-Cys was conjugated to the ω-aldehyde PVP and the conjugation efficiency was compared to our PBLG-Cys block. In the case of PBLG-b- Cys the in situ deprotection and conjugation as well as a two-step deprotection and conjugation reaction with PVP resulted in very low conjugation efficiency. The cysteine end-functional PBLG resulted in near quantitative conjugation with PVP. The critical micelle concentration (CMC) for PVP90-b-PBLG54 was determined to be 6 μg/mL, using fluorescence spectroscopy. Particle sizes were determined with TEM and DLS and found to range from 25 nm to 120 nm depending on the polymer block lengths as well as hydrophobic/hydrophilic block length ratios. Furthermore, when the micelles were subjected to an increased acidic environment, the labile benzyl ester bonds were hydrolyzed. This was observed with TEM where the particle sizes increased 10-fold to form vesicular structures. Hydrolysis was further confirmed with ATR-FTIR and 1H-NMR spectroscopy. Cytotoxicity tests confirmed that the copolymer micelles had good cell compatibility at high concentrations such as 0.9 mg/mL. Investigation into drug loading using a pyrene probe confirmed the viability of using PVP-b-PBLG as a responsive DDS. / AFRIKAANSE OPSOMMING: Die kombinasie van natuurlike en sintetiese polimere maak dit moontlik vir die sintese van gevorderde hibried kopolimere. Hierdie kopolimere het aanwending in biomediese gebiede, een so 'n gebied is in medisinale vervoer sisteme (MVS). 'n Modulêre benadering is in hierdie studie gebruik om amfifiliese blok kopolimere te berei. Omkeerbare addisie-fragmentasie kettingoordrag (OAFO) is gebruik as die sintetiese tegniek vir die polimerisasie van N-vinielpirolidoon (NVP). OAFO is 'n veelsydige metode om polimere te berei met beheer oor molekulêre gewig en dispersiteit (Đ). 'n Xantaat kettingoordrag agent (KOA) is gebruik om die hidrofiliese poli(N-vinielpirolidoon) (PVP) blok te sintetiseer. ‘n Aldehied endgroep was deur die terminale xantaat funksionaliteit berei, ‘n proses wat geoptimiseer is tot kwantitatiewe omsetting. 'n Di-xantaat KOA is gesintetiseer om, na modifikasie, 'n α, ω-telecheliese polimeer te produseer. Die polipeptied was gesintetiseer deur middel van ’n ringopening polimerisasie van Nkarboksianhidriede (ROP NKA). Die lewende en beheerbare ROP van NKAe is 'n metode wat lei tot polipeptiede sonder ’n gedefinieerde aminosuur volgorde. Poli(γ- benzyl-L-glutamaat) met 'n lae dispersiteit (Đ = 1.10 – 1.15), is gesintetiseer deur gebruik te maak van kondisies wat die behoud van 'n terminale primêre amien bevorder. 'n Beskermde sistien-funksionaliteit is ingebou via die terminale amien met behulp van peptiedsintese tegnieke. Die tiol beskerming van die asetamidometiel (Asm) groep kon nie gekleef word deur gebruik te maak van tradisionele metodes nie, maar ‘n nuwe proses is ontwikkel om die Asm groep te kleef sowel as om die hidrolise van die bensiel esters te inhibeer. Die koppelings reaksie het effektief verloop in DMF by 'n effens verhoogde temperatuur en sodoende is die amfifiliese hibried blok-kopolimere, PVP-b-PBLG berei. Twee verskillende PBLG kettings is gekoppel aan die ω-aldehied PVP en die koppeling doeltreffendheid is vergelyk. Daar is bevind dat net die sistien end-funksionele PBLG tot kwantitatiewe konjugasie kon lei. Die kritiese misel konsentrasie is bepaal vir PVP90-b-PBLG54 as 6 μg/mL met behulp van fluoressensie spektroskopie. Die deeltjie-groottes is bepaal met TEM en DLS en wissel van 25 nm tot 120 nm, afhangende van die polimeer bloklengtes sowel as hidrofobiese / hidrofiliese blok lengte verhoudings. Die miselle is blootgestel aan 'n verhoogde suur omgewing, wat tot die hidrolise van die bensiel ester groepe gelei het. TEM het getoon dat die deeltjie-groottes met 10-voud vergroot het tot vesikulêre strukture. Hidrolise is verder bevestig met ATR-FTIR en 1H-KMR spektroskopie. Sitotoksiese toetse het bevestig dat die miselle geen of min toksisiteit toon teenoor eukariotiese selle nie, selfs teen 'n hoë konsentrasies soos 0.9 mg/ml. Die medisinale behoud vermoë is met behulp van pireen bevestig en dus ook die potensiaal van PVP-b-PBLG as ‘n moontlike MVS.

A doença periodontal é uma patogenia que afeta as estruturas de suporte dos dentes com formação da bolsa periodontal, e caso não tratada, em estágios mais avançados o periodonto é destruído, ocasionando perda do dente. O tratamento concerne em duas etapas, à remoção mecânica do biofilme e cálculo dentário por raspagem e alisamento radicular e utilização de antimicrobianos. Ainda é comum a utilização de antimicrobiano de ação sistêmica, embora, tenha eficácia reduzida e frequentes efeitos adversos. Em face disto, justifica-se o desenvolvimento de sistemas de liberação sustentada de fármaco intrabolsa periodontal a fim de sanar os inconvenientes da terapia sistêmica. Contudo, aspectos como espaço anatomofisiológico e fluído gengival crevicular intrínsecos à bolsa, são limitações pertinentes durante o desenvolvimento. Portanto, este trabalho teve como objetivo desenvolver sistemas e formulações precursoras de fase líquido cristalina baseados em monolinoleato de glicerila (MLG) e cremophor (CREM) com gelificação in situ em fase líquido cristalina. Por meio de planejamento fatorial 32 foram obtidos 9 sistemas sem adição de fármaco e 9 formulações contendo metronidazol com diferentes razões de MLG/CREM e %H2O. Quando caracterizados por microscopia de luz polarizada e espalhamento de raios-X a baixo ângulo, os sistemas e formulações com menor razão MLG/CREM e conteúdos de água de 5, 10 e 15% apresentaram organização micelar isotrópica. Eles também apresentaram rápida erosão no ensaio de captação de água, inviabilizando sua transição para mesofase. Os sistemas e formulações com média e alta razão MLG/CREM e conteúdos de água de 5, 10 e 15% apresentaram coexistência de fases em transição líquido cristalina. Apresentaram rápida captação de água com transição para fase cúbica. Contudo, com exceção do sistema e formulação com alta razão MGL/CREM e 15% de água, os demais sistemas e formulações, apresentaram propriedades de fluxo ideais para fácil aplicação por seringa acoplada a agulha, e comportamento viscoelástico adequado para promover espalhabilidade e retenção no interior da bolsa. Os sistemas e formulações com média e alta razão de MLG/CREM apresentaram melhor mucoadesão. Dentre as formulações, apenas a com média razão de GML/CREM e 5% de água, apresentou perfil bi-modal de liberação mantendo concentrações de metronidazol acima do MIC por 6 dias, característica desejável para sistemas de liberação intrabolsa periodontal. Portanto, todos os resultados advogam a favor desta formulação como potencial candidata ao emprego clínico como sistema de liberação de fármaco intrabolsa periodontal. / Periodontal disease is a chronic infection that affects the structural supports of the tooth with periodontal pocket formation. If untreated, the periodontium may be destroyed, which can cause the tooth loss in the latest stages of the disease. The treatment for this infection comprises two stages, the mechanical removal of plaque and calculus by scaling and root planing and the use of antimicrobials. It is still common to use systemic antimicrobials, even though it has a reduced efficacy and frequent adverse effects. For the reason, justified the development of systems for sustained release of drug intra-pocket periodontal in order to solve the drawbacks of systemic therapy. The main limitations for the development of these systems are the anatomical and physiological aspects of the periodontal pocket, which includes the limited size and the depth area and the natural irrigate flow of the gingival crevice fluid (GCF). So, this study aimed to develop precursor liquid crystalline systems and formulations based on glyceryl monolinoleate (GML) and cremophor (CREM), with in situ gelation to liquid crystalline phases. Through a 32 factorial design 9 systems without drug and 9 formulations containing metronidazole were obtained. Different ratios of GML / CREM and water percentage were evaluated. When characterized by Small-Angle X-ray Scattering and Polarized Light Microscopy, lower ratio GML / CREM with 5, 10 and 15% H2O produce isotropic micellar organization. They also showed high erosion during water uptake, thus, precluding in situ crystalline transition. Medium and high ratio GML / CREM with 5, 10 and 15% H2O indicated the coexistence of regions in transition to the crystalline phase. They showed characteristic peaks of cubic phases, rapid water uptake, higher viscosity and increased mucoadhesion. Moreover, with the exception of the formulation containing high ratio and high water content, other systems and formulations with medium ratio GML / CREM and 5, 10, 15% of water and high ratio and 5 and 10% of water had flow properties ideal for easy application through needle fitted syringes, viscoelastic behavior appropriate to promote spreadability and might favor retention within the periodontal pocket. However, only the formulation with higher ratio and the lowest water content presented a bimodal release profile of metronidazole, maintaining concentrations above the MIC for 5 days, with the release of 100% of the drug added to the formulation. These are desirable features for delivery systems designed for periodontal pockets. Therefore, all results advocate in favor of the formulation with high ratio of GML / CREM and 5% of water as a potential candidate for clinical use as drug delivery system for the periodontal pocket.

伴隨著納米科技的發展，越來越多的納米顆粒系統已經應用於生物醫學领域。其中，二氧化硅納米顆粒因其簡單易操作的表面化學性質和在生理環境中的良好穩定性，已被廣泛認為是最有前途的治療和診斷載體之一。 / 在本論文中，我首先對二氧化硅納米顆粒與人類細胞之間的作用進行了系統研究。這些作用包括了以下主要特點: 胞吞和胞吐被分別確定為納米顆粒主要的進入和離開細胞的主要途徑; 大部份的納米顆粒被發現存在於有膜結構的細胞器里，這些細胞器相當穩定（不易破損），只有很少的一部份納米顆粒被釋放到細胞質里; 納米顆粒和細胞之間的作用是動態的，它們進入細胞內的數量由其在細胞培養液中的數量和形態（聚集的程度）所決定。正是這些特點決定了二氧化硅顆粒在低濃度時的低細胞毒性。 / 緊接著我比較了兩種最常見的二氧化硅納米顆粒（晶體態和無定型態）引入細胞后對細胞所帶來的影響。儘管兩種形態的納米顆粒所造成的細胞毒性都比較低，但是更細緻的分析揭示了它們對細胞及其衍化途徑的不同影響。細胞吞入晶體態的二氧化硅納米顆粒后，其內部的活性氧物質含量顯著提高，這種變化會導致細胞線粒體功能受損（表現為線粒體增生）並且最終將細胞導向死亡。不過只有在p53基因缺失的細胞中才有這種由活性氧物質水平升高導致的細胞損傷，p53正常的細胞卻能抵禦這種來自晶體態二氧化硅納米顆粒的刺激。而無定型態二氧化硅納米顆粒對生物系統無損害，因而有發展為藥物載體的巨大潛力。 / 基於對二氧化硅顆粒細胞毒性研究的理解，我們設計了一種新型納米載體--金核/二氧化硅殼層（Au@SiO₂）納米顆粒用於藥物輸運。在這一體系中，無定形態二氧化硅和金納米顆粒的優勢被整合在一起，同時光敏劑（PS）藥物分子被裝載在二氧化硅殼層內。對比於自由形式的PS，裝載在Au@SiO₂納米顆粒中的PS展示出增強的藥效。需要強調的是，用這種納米顆粒處理的細胞以阻梗壞死為主要的死亡途徑，代替了凋亡這種不太有效的方式。在光照下，金的等離子體效應被發現能促進PS的光響應過程，這使得細胞殺死率得到了大幅度增強。這一效應得益于我們把PS束縛在金核的表面，同時保證金表面等離子體振盪能量和PS吸收能量的配對。此外，把PS裝載在二氧化硅中會引起PS有益的光化學改變。這些作用結合在一起導致了藥效的提高。這些機理能被普遍應用於納米顆粒裝載藥物分子的設計中，為最優化設計提供指導。 / With recent development of nanotechnology, various nanoparticulate systems have been proposed to serve as functional units for biomedical applications in many innovative ways. Among various possible choices, silica nanoparticles (NPs) enjoys easily modifiable surface chemical characteristics and excellent stability in physiological environment. Therefore, it is considered as one of the most promising carrier candidate for therapeutic and diagnostic applications. / A systematic study on the interaction between silica nanoparticles and human cells is first carried out in the present thesis work. Endocytosis and exocytosis are identified as major pathways for NPs entering, and exiting the cells, respectively. Most of the NPs are found to be enclosed in membrane bounded organelles, which are fairly stable (against rupture) as very few NPs are released into the cytoplasma. The nanoparticle-cell interaction is a dynamic process, and the amount of NPs inside the cells is affected by both the amount and morphology (degree of aggregation) of NPs in the medium. These interaction characteristics determine the low cytotoxicity of SiO₂ NPs at low feeding concentration. / Experiments were then designed to compare the the biological consequence of two most common form of SiO₂ nanoparticles, i.e., crystalline and amorphous NPs, when they were introduced to human cells. Although the apparent cytotoxicity of both types of NPs seems to be low, more detailed characterizations disclose the profound difference induced by the crystalline and amorphous ones, resulting in significantly different cell evolution pathways. Crystalline NPs but not amorphous ones are found to drastically increase the recative oxygen species (ROS) level in the cells, which can cause mitochondria dysfunction (being expressed as mitochondria proliferation), and eventually direct the cell into apoptosis. Nonetheless, only p53 deficient cells are subjective to such ROS induced cell damage, while p53 proficient cells can accommodate the stimulation from crystalline SiO₂ NPs. The amorphous SiO₂ NPs are found to be benign in the biological systems, and have great potential to be developed as nanomedicine. / Base on the understanding obtained from the toxicology study of the SiO₂ NPs, we have designed a special nanocarrier system for drug delivery. We have combined advantages of both SiO₂ and Au NPs by constructing Au-core/SiO₂-shell (Au@SiO₂) nanocarriers with the photosensitizer (PS) drug embedded in the SiO₂ shell layer. Compared with free PS, PS loading in the Au@SiO₂ NPs showes a enhanced drug efficacy. In particular, the cells treated with the NP drug take necrosis as a major death path instead of apoptosis, which is a much less effective route. The Au plasmonic effect is found to promote the photo-response of the PS drug under light irradiation, contributing to the largely decreased cell viability. Nevertheless, one shall note that spatial confinement of the drug moledules to the close proximity of the Au core and an energy match between the drug absorption and the Au surface plasmon resonance are critical in manifesting the plasmonic effect. At the same time, embedding the drug in the SiO₂ matrix leads to favorable change in the photochemical process. The combined effects brought by the Au@ SiO₂ NP carrier is responsible for the high drug efficacy. These mechanisms can be generally valid in engineering drug molecule incorporation into NP carriers and also give guidance for the optimum design of the NP drug carrier. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chu, Zhiqin = 二氧化硅納米顆粒與人類細胞的作用及其在生物醫學方面的應用 / 褚智勤. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 120-137). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chu, Zhiqin = Er yang hua gui na mi ke li yu ren lei xi bao de zuo yong ji qi zai sheng wu yi xue fang mian de ying yong / Chu Zhiqin. / Table of contents --- p.VIII / List of figures --- p.XIII / List of tables --- p.XIX / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Background --- p.4 / Chapter 2.1 --- Overview of the silica-based nanoparticles for bio-medical applications --- p.4 / Chapter 2.2 --- Health issue on the silica-base nanoparticles --- p.5 / Chapter 2.3 --- Understanding the nano-bio interface --- p.6 / Chapter 2.3.1 --- Nano-bio interface in vitro --- p.7 / Chapter 2.3.2 --- Nano-bio interface in vivo --- p.10 / Chapter 2.4 --- Bio-application of silica-based nanoparticles --- p.11 / Chapter 2.4.1 --- Use of silica nanoparticle as imaging agent --- p.11 / Chapter 2.4.2 --- Use of silica nanoparticle as drug carrier --- p.12 / Chapter 2.4.3 --- Use of silica nanoparticle as coating media --- p.12 / Chapter 2.5 --- Surface plasmon of gold nanostructures and its bio-application --- p.13 / Chapter 2.5.1 --- Introduction to the SPR of gold nanostructures --- p.13 / Chapter 2.5.2 --- Synthesis of gold NRs and their SPR effect --- p.13 / Chapter 2.5.3 --- SPR of gold NRs in bio-application --- p.16 / Chapter Chapter 3 --- Experimental --- p.18 / Chapter 3.1 --- Standard methodologies for nanoparticle preparation and their feeding to the cells --- p.18 / Chapter 3.2 --- Cell sampling for room temperature TEM study --- p.18 / Chapter 3.3 --- Developing methods to distinguish NPs in cell sample under TEM --- p.20 / Chapter 3.4 --- Confocal microscopy study --- p.21 / Chapter 3.4.1 --- Study the photoluminescence of various dye molecules --- p.21 / Chapter 3.4.2 --- Study the two photon luminescence (TPL) of Au NRs --- p.23 / Chapter 3.5 --- UV-Vis-NIR spectrophotometer and fluorescence spectrophotometer --- p.25 / Chapter 3.6 --- Flow-cytometry --- p.26 / Chapter 3.7 --- Western plot --- p.28 / Chapter 3.8 --- Colormetric assays and other biological labels --- p.28 / Chapter 3.8.1 --- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test --- p.28 / Chapter 3.8.2 --- Mitochondria, lysosome and nucleus staining --- p.30 / Chapter 3.8.3 --- Detection of apoptosis --- p.31 / Chapter 3.8.4 --- Detection of various reactive oxygen species (ROS) --- p.31 / Chapter Chapter 4 --- Silica NPs interact with human cells --- p.34 / Chapter 4.1 --- Introduction --- p.34 / Chapter 4.2 --- Characterization of silica nanoparticles --- p.36 / Chapter 4.3 --- General description of the NPs’ uptaking and excreting process --- p.42 / Chapter 4.4 --- Tracking of NPs inside the cells --- p.53 / Chapter 4.5 --- Factors influencing the NP-cell interaction and exocytosis process --- p.55 / Chapter 4.5.1 --- The effect of serum (in the incubation medium) on cellular uptake --- p.55 / Chapter 4.5.2 --- Crystallinity effectdistribution of amorphous and crystalline SiO₂ NPs in the cells --- p.57 / Chapter 4.5.3 --- Factors affecting the exocytosis process --- p.59 / Chapter 4.6 --- Cytotoxic effect of silica NPs --- p.60 / Chapter 4.7 --- Conclusion --- p.63 / Chapter Chapter 5 --- Genotoxic effect specifically induced by crystalline SiO₂ nanoparticles in p-53 deficient human cells --- p.65 / Chapter 5.1 --- Introduction --- p.65 / Chapter 5.2 --- The difference between crystalline and amorphous silica NPs --- p.66 / Chapter 5.2.1 --- Mitochondria multiplication specially induced by crystalline silica NPs --- p.68 / Chapter 5.2.2 --- DNA fragmentation specially observed in crystalline silica NPs treated cells --- p.71 / Chapter 5.3 --- The cell line sensitive cytotoxicity of crystalline silica NPs --- p.79 / Chapter 5.3.1 --- A general phenomenon of mitochondria increase in p-53 negative cell lines --- p.80 / Chapter 5.3.2 --- General biological consequence of such mitochondria increase --- p.82 / Chapter 5.4 --- Conclusion --- p.83 / Chapter Chapter 6 --- Surface plasmon enhanced drug efficacy for PDT using core shell Au@SiO₂ nanoparticle carrier --- p.84 / Chapter 6.1 --- Introduction --- p.84 / Chapter 6.1.1 --- Brief introduction to the photodynamic therapy (PDT) and photosensitizer (PS) --- p.84 / Chapter 6.1.2 --- Brief introduction to the SPR enhanced generation of ROS --- p.86 / Chapter 6.2 --- Using Au@SiO₂ NPs as drug carrier --- p.88 / Chapter 6.2.1 --- Growth of gold NRs and their controllable oxidation --- p.88 / Chapter 6.2.2 --- Preparation and characterization of Au@(SiO₂-MB) NPs --- p.90 / Chapter 6.2.3 --- Confirmation of MB loading into silica shell --- p.92 / Chapter 6.3 --- Enhanced PDT drug (MB) efficacy when loaded in Au@SiO₂ NPs --- p.95 / Chapter 6.3.1 --- Cellular uptake pathway of free MB and Au@SiO₂ NPs --- p.95 / Chapter 6.3.2 --- Comparing the efficacy of free MB, SiO₂-MB NPs and Au@(SiO₂MB) NPs --- p.98 / Chapter 6.4 --- Studying the behavior of free MB and Au@(SiO₂-MB) NPs as PDT agent --- p.100 / Chapter 6.4.1 --- Comparing the ability of generating ROS by free MB and Au@(SiO₂MB) NPs --- p.100 / Chapter 6.4.2. --- Comparing the types of ROS generated by free MB and Au@(SiO₂MB) NPs --- p.103 / Chapter 6.4.3 --- Comparing the cellular death pathway triggered by free MB and Au@(SiO₂-MB) NPs --- p.105 / Chapter 6.5. --- Discussion on the mechanism for the enhanced efficacy --- p.109 / Chapter 6.5.1 --- Excluding the photothermal effect of Au NRs core --- p.109 / Chapter 6.5.2 --- The role of SiO₂ in the Au@SiO₂ NPs carrier --- p.111 / Chapter 6.5.3 --- Attributing the enhanced efficacy to plasmonic effect of Au NRs core --- p.112 / Chapter 6.6 --- Exploring the potential of using Au@SiO₂ NP carrier in vivo --- p.114 / Chapter 6.7 --- Conclusion --- p.116 / Chapter Chapter 7 --- Conclusion --- p.118 / References --- p.120