Tesis sobre el tema "Multidrug nanoparticles"

L'ischémie/ reperfusion cardiaque est une condition pathologique complexe impliquant divers mécanismes de signalisation. En raison de leurs interactions, les traitements disponibles pour les lésions de reperfusion sont souvent insuffisants. Il est donc crucial d'explorer des approches intégrantes plusieurs agents ciblant différentes voies biologiques. Bien que la nanomédecine offre des opportunités pour protéger les principes actifs et optimiser le ciblage thérapeutique, elle fait face à des défis tels qu'un faible taux d'incorporation des médicaments. La méthode de squalenoylisation pourrait résoudre ces problèmes. Nous avons d'abord étudié in vitro les nanoparticules à base de squalène contenant de la cyclosporine A (SqCsA), en évaluant leur toxicité et capacité cardioprotective. Nous avons ensuite testé des formulations de squalène-adénosine (SQAd) dans un modèle murin in vivo, observant des effets bénéfiques sur la fonction cardiaque, bien que sans atteindre une signification statistique. Par la suite, nous avons développé des nanoparticules SQAd/Vit E, testées dans un modèle rat amélioré d'ischémiereperfusion myocardique, et utilisé des méthodes d'analyse approfondies pour capturer des effets cardioprotecteurs détaillés. Enfin, nous avons étudié la formation de la couronne protéique sur ces nanoparticules afin d'éclairer leur comportement in vivo dans les fluides biologiques. Ces travaux soulignent le potentiel des nouvelles formulations nanomédicales dans le traitement des lésions d'I/R cardiaques
Myocardial ischemia/ reperfusion is a complex pathological condition involving various signaling mechanisms. Due to their interactions, available treatments for reperfusion injuries often prove insufficient. It is crucial to explore approaches that integrate multiple agents targeting different biological pathways. While nanomedicine offers opportunities to protect active ingredients and optimize therapeutic targeting, it faces challenges like low drug incorporation rates. The squalenoylation method holds potential to address these issues. We first studied squalene-based NPs containing cyclosporine A (SqCsA) in vitro, assessing their toxicity and cardioprotective capacity. We then tested squalene-adenosine formulations (SQAd) in an in vivo mouse model, observing beneficial effects on cardiac function, though statistical significance was not reached. Subsequently, we developed SQAd/Vit E nanoparticles and tested them in an optimized rat model of myocardial ischemia-reperfusion, applying comprehensive analysis methods and captured detailed cardioprotective effects. Finally, we investigated protein corona formation on these nanoparticles to shed light on their in vivo behavior in biological fluids. These findings highlight the potential of novel nanomedical formulations in treating cardiac I/R injuries

Philosophiae Doctor - PhD
Ovarian cancer is called the “Silent Killer” as it is often diagnosed in advanced stages of the disease or misdiagnosed which ends with a poor prognostic outcome for the patient. A high rate of disease relapse, a high incidence-to-mortality ratio as well as acquired multidrug resistance makes it necessary to find alternative diagnostic- and therapeutic tools for ovarian cancer. Nanotechnology describes molecular devices with at least one dimension in the sub- 1μm scale and has been suggested as a possible solution for overcoming challenges in cancer multidrug resistance as well as early diagnosis of the disease. One-pot synthesized gold nanoparticles were used to demonstrate in vitro drug delivery of doxorubicin in a manner which overcame the cytoprotective mechanisms of a multidrug resistant ovarian carcinoma cell line (A2780cis) by inducing apoptosis mediated by caspase-3 within 3h of treatment. The gold nanoparticles were further functionalized with nitrilotriacetic acid and displayed specific interaction with a 6xHis-tagged cancer targeting peptide, chlorotoxin. Proprietary indium based quantum dots were functionalized with the same surface chemistry used for gold nanoparticles and bioconjugated with chlorotoxin. Wide field fluorescence studies showed the peptide-quantum dot construct specifically targeted enhanced green fluorescent tagged matrix metalloproteinase-2 transfected A2780cis cells in a specific manner. The cytoprotective multidrug resistant mechanisms of the ovarian carcinoma was overcome successfully with a single dose of doxorubicin loaded gold nanoparticles and tumour specific targeting was demonstrated using quantum dots with a similar surface chemistry used for the gold nanoparticles.

Cancer is a group of diseases in which normal cells are converted to cells capable of autonomous growth and invasion. In the chemotherapeutic control of cancer, drugs are usually given systemically so they reach toxic levels in healthy cells as well as cancer cells. This causes serious side effects. Another important problem with chemotherapy is resistance developed to cytotoxic drugs (multi drug resistance). Doxorubicin (Dox) occupies a central position in the treatment of breast cancer. However doxorubicin induced cardiac toxicity is associated with a high incidence of morbidity and mortality. Resistance of malignant tumors to Dox is another important cause of treatment failure in patients with cancer. One approach to overcome Dox-related toxicity is to use polymeric drug carriers, which direct the Dox away from heart tissue, and allow usage of lower dosages. In this present study two different anti-cancer drug delivery methods were evaluated. Dox was encapsulated in PLGA microparticles by single and double microemulsion solvent evaporation techniques. The highest entrapment of doxorubicin within PLGA microspheres obtained by optimization of process parameters. A sustained release of doxorubicin was obtained for 20 days. Several protein transduction domains are known to have the ability to pass through biological membranes. One such peptide is HIV-1 TAT. In this study TAT was evaluated for its ability to carry Dox into Dox resistant MCF-7 tumor cells. Dox peptide conjugate was more potent than free drug. The concentration of drug in resistant cancer cells was increased indicating a partial reversal of drug resistance.

One of the major mechanisms of multidrug resistance involves an efflux protein, P-glycoprotein (Pgp), which pumps commonly used anticancer drugs such as taxanes out of cells, leading to a decrease in cellular drug accumulation. The overall goal of this project was to develop strategies to enhance intracellular drug accumulation and cytotoxicity of nanoparticulate taxanes in multi-drug resistant (MDR) cell lines. Paclitaxel (PTX) loaded nanoparticles fabricated from micelle forming MePEG₁₁₄-b-PCL₁₉ and nanosphere forming MePEG₁₁₄-b-PCL₁₀₄ were compared for drug and block copolymer uptake, and cytotoxicity in drug sensitive MDCKII and drug resistant MDCKII-MDR1 cell lines. PTX loaded micelles were more cytotoxic than PTX loaded nanospheres. Co-administration of the known Pgp inhibitor, MePEG₁₇-b-PCL₅, with PTX loaded micelles or nanospheres significantly increased drug cytotoxicity in MDCKII-MDR1 cells. Mixed molecular weight (MW) PCL₂₀₀/PCL₅ nanoparticles composed of long hydrophobic block, MePEG₁₁₄-b-PCL₂₀₀, and MePEG₁₇-b-PCL₅, were developed and characterized for the co-delivery of taxanes and Pgp inhibitor. Both PTX and docetaxel (DTX) loaded mixed MW PCL₂₀₀/PCL₅ nanoparticles were demonstrated to release MePEG₁₇-b-PCL₅ in a controlled release manner and increase drug cytotoxicity in MDR cells as compared to the drug loaded MePEG₁₁₄-b-PCL₂₀₀ nanoparticles in the absence of MePEG₁₇-b-PCL₅. The mixed MW nanoparticles remained in the plasma for longer than the drugs with approximately 3% of the injected dose remaining 24 hrs post injection. Ultrasound irradiation was investigated as a potential strategy to enhance the cytotoxicity of PTX loaded MePEG-b-PDLLA micelles in MDR cells. Using an ultrasound regime of a single 10-second burst of high frequency (4 MHz) and high intensity (32 W/cm²) ultrasound, it was shown that ultrasound irradiation resulted in a two-fold increase in intracellular uptake of PTX in drug sensitive MDCKII and MCF-7 cell lines and their respective Pgp-overexpressing MDCKII-MDR1 and NCI-ADR counterparts as compared to untreated cells (no ultrasound). The enhanced accumulation and retention of PTX resulting from ultrasound treatment translated into greater cytotoxicity in both drug sensitive and resistant cell lines. In conclusion, we have demonstrated two promising strategies for enhancing MDR cellular drug accumulation and effectiveness: the use of mixed molecular weight taxane loaded nanoparticles and ultrasound irradiation.
Pharmaceutical Sciences, Faculty of
Graduate

Philosophiae Doctor - PhD (Chemistry)
Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (MTB) that usually affects the lungs leading to severe coughing, fever and chest pains. It was estimated that over 9.6 million people worldwide developed TB and 1.5 million died from the infectious disease of which 12 % were co-infected with human immunodeficiency virus (HIV) in the year 2015. In 2016 the statistics increased to a total of 1.7 million people reportedly died from TB with an estimated 10.4 million new cases of TB diagnosed worldwide. The development of the efficient point-of-care systems that are ultra-sensitive, cheap and readily available is essential in order to address and control the spread of the tuberculosis (TB) disease and multidrugresistant tuberculosis.

Les syndromes inflammatoires, marqués par une réaction excessive du système immunitaire, sont associés à de nombreuses conditions pathologiques comme le sepsis, les accidents d’ischémie/reperfusion ou l’athérosclérose. En effet, une réponse inflammatoire excessive à l’encontre des tissus caractérise souvent l’apparition et la pathophysiologie de ces maladies complexes pour lesquelles ils manquent toujours des traitements efficaces. Ainsi, la plupart des thérapies disponibles pour adapter la réponse inflammatoire sont limitées la plupart du temps par des effets secondaires indésirables, une mauvaise spécificité et par l’apparition d’états d’immunodéficience.Des avancées en délivrance de médicaments ont permis le développement de nombreux outils qui aident à adapter la stratégie d’administration d’agents pharmaceutiques. Notamment, les “nanomédecines” ont permis d’obtenir de nombreuses innovations en oncologie et en technologie diagnostique. En améliorant le ciblage des médicaments et en protégeant l’agent pharmaceutique d’une métabolisation précoce, les nanomedicines permettent d’améliorer l’index thérapeutique de certaines molécules, résultant en une amélioration du prognostic patient. Néanmoins, avec ces promesses viennent des limitations notables, comme le faible taux de chargement en drogue de certaines nanoformulations, un développement industriel compliqué ou un mauvais contrôle du relargage.Pour répondre à ces limitations, l’équipe du Pr. Couvreur développe depuis 2004 une nouvelle stratégie d’encapsulation des médicaments, appelée « squalenoylisation ». Ainsi, le couplage d’agents actifs avec des dérivés du squalène, un lipide endogène, biocompatible et biodégradable, permet d’obtenir des nanoparticules stables, avec haut taux de chargement et faible toxicité. Les nanoparticules obtenues présentent dans de nombreux cas une pharmacocinétique plus avantageuses ainsi que distribution plus ciblée aux zones pathologique. L’application de cette technique à l’adénosine, une molécule au potentiel thérapeutique important mais limité par un temps de demi-vie plasmatique extrêmement court, a fourni des résultats particulièrement prometteurs dans le cadre du traitement de l’ischémie cérébrale et du traumatisme de la moëlle épinière.Un autre avantage des nanoparticules à base de squalène est qu’elles rendent possible l’encapsulation de plusieurs agents thérapeutiques au sein d’un même système, permettant ainsi des traitements multi-drogue. Ceci est un outil important dans le contexte d’une réponse excessive du système immunitaire, où convergent souvent plusieurs facteurs pour faire progresser la maladie. Par exemple, il a été établi que l’auto-inflammation est souvent maintenue par des cercles vicieux entre stress oxydant et cascades pro-inflammatoires, ces deux voies de signalisation contribuant l’une à l’autre et inhibant le retour à l’homéostasie. Il n’existe pour l’instant pas de thérapies efficaces contre ce couplage pathologique. Part conséquent, un des objectifs de cette thèse était de développer et de tester sur de modèles précliniques d’inflammation excessive, des nanoparticules à base de squalène encapsulant deux agents thérapeutiques : l’adénosine, comme médiateur endogène des réponses inflammatoires et un antioxydant comme inhibiteur du stress oxydant. Notre hypothèse étant qu’une thérapie multi-drogue pourrait être avantageuse pour contrer les nombreux processus pathogènes qui se renforcent mutuellement lors des réponses inflammatoires ; mais aussi qu’une formulation sous forme de nanoparticules pourrait fournir des propriétés de ciblage intéressantes
Advances in drug delivery have led to the development of many tools that help tailor the drug delivery strategy. In particular, “nanomedicines” have made it possible to obtain numerous innovations in oncology and diagnostic technology. By improving drug targeting and protecting the pharmaceutical agent from early metabolism, nanomedicines improve the therapeutic index of certain molecules, resulting in improved patient prognosis. However, with these promises come notable limitations, such as the low drug loading rate of certain nanoformulations, complicated industrial development or poor release control. Squalene-based nanoparticles have been developed to meet these limitations. Another advantage of squalene-based nanoparticles is that they make it possible to encapsulate several therapeutic agents within the same system, thus allowing multi-drug treatments.This is an important tool in the context of an excessive inflammatory response, where many factors often converge to advance the disease. Therefore, one of the objectives of this thesis was to develop and test on preclinical models of inflammation, squalene-based nanoparticles encapsulating two therapeutic agents: adenosine, as an endogenous mediator of inflammatory responses and an antioxidant as an inhibitor of oxidative stress. Our hypothesis is that a multi-drug therapy could be advantageous to counter the many pathogenic processes which reinforce each other during inflammatory responses, but also that a formulation in the form of nanoparticles could provide interesting targeting properties. During the work of this thesis, we also have the feasibility of industrial translation of the synthesis of squalene-based bioconjugates

Arranjos supramoleculares combinando o lípide catiônico brometo de dioctadecildimetilamônio (DOD) com polímeros, como carboximetilcelulose (CMC) e cloreto de poli(dialildimetilamônio) (PDDA), foram preparados na forma de nanopartículas (NPs), na ausência ou presença de antimicrobiano tradicional, como a claritromicina (CLA). NPs preparadas por atração eletrostática entre os fragmentos de bicamada (BF) de DOD, CMC e PDDA foram avaliadas, in vitro, quanto à atividade contra isolados clínicos de micro-organismos multirresistentes (MR) a antimicrobianos, como Pseudomonas aeruginosa MR, Klebsiella pneumoniae produtora da enzima carbapenemase do tipo KPC, Staphylococcus aureus resistente à meticilina/oxacilina (MRSA) e Candida albicans resistente ao fluconazol, através do método de plaqueamento e contagem de viáveis. As NPs de DOD BF/CMC/PDDA apresentam alta atividade de amplo espectro contra micro-organismos MR, em que o PDDA é o componente responsável pela excelente atividade biocida das NPs. O mecanismo de ação antimicrobiana indica a dissociação dessas NPs na presença dos micro-organismos, com a remoção de biopolímeros da parede celular microbiana pelo PDDA, conforme visualizado por microscopia eletrônica de varredura, ocorrendo lise da membrana microbiana e liberação de compostos fosforilados para o meio extracelular. Também foram desenvolvidas neste trabalho NPs carreadoras de CLA à base de DOD e polímeros. Solução etanólica contendo CLA/DOD foi injetada em solução aquosa de CMC, formando arranjos coloidalmente estáveis e aniônicos, que posteriormente foram adicionados de solução de PDDA, para a obtenção de arranjos estáveis e catiônicos. CLA/DOD/CMC e CLA/DOD/CMC/PDDA NPs incorporaram CLA em quantidade suficiente para inibir o crescimento de M. abscessus no interior de macrófagos bem como evitar a formação de biofilmes, sendo que altas doses de CLA foram tóxicas aos macrófagos, enquanto doses menores apresentaram baixa toxicidade e boa atividade antimicrobiana. NPs catiônicas carreando CLA foram tóxicas aos macrófagos nas concentrações de PDDA testadas. A natureza particulada das CLA NPs possivelmente aumenta a retenção intracelular de CLA em comparação com CLA livre, podendo prolongar atividade da CLA contra patógenos intracelulares. Desta maneira, arranjos supramoleculares combinando lípide e polímeros, com ou sem antimicrobianos tradicionais poderão encontrar diversas aplicações nas áreas farmacêutica, médica, alimentícia e biotecnológica.
Supramolecular assemblies combining cationic lipid dioctadecyldimethylammonium bromide (DOD) and polymers, such as sodium carboxymethylcellulose (CMC) and poly(diallyldimethylammonium chloride) (PDDA), were prepared as nanoparticles (NPs), in the absence or presence of traditional antibiotic, such as clarithromycin (CLA). NPs prepared by electrostatic attraction between DOD bilayer fragments (BF), CMC and PDDA were evaluated against clinical strains of multidrug resistant (MDR) microorganisms, such as Pseudomonas aeruginosa MDR, Klebsiella pneumoniae producer of KPC carbapenemase enzyme, methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans fluconazole resistant, by plating and colony forming unities counting. DOD BF/CMC/PDDA NPs display high and broad-spectrum activity against MDR microrganisms, and PDDA is the excellent biocidal component in the NPs. The mechanism of antimicrobial action shows that NPs disassembly in the presence of microrganisms, with biopolymers withdrawn from the cell wall, as observed by scanning electron microscopy, consecutively lysing bacterial membrane as determined from the leakage of inner phosphorylated compounds. In this work there have also been developed NPs, based on lipid and polymers, as carriers for CLA. Ethanolic solution co-solubilizing CLA/DOD was injected in CMC aqueous solution, yielding colloidaly stable and anionic NPs, that were further added of PDDA solution, yielding stable and cationic NPs. CLA/DOD/CMC NPs and CLA/DOD/CMC/PDDA NPs incorporated CLA at doses high enough to inhibit M. abscessus growth inside macrophages or in biofilms. Larger CLA doses were toxic to macrophages while lower CLA doses reduced toxicity to macrophages despite their high antimicrobial activity. Cationic CLA NPs exhibited substantial toxicity against macrophages at the PDDA concentrations tested. The particulate nature of these CLA NPs possibly increases intracellular CLA retention in comparison to free CLA, probably extending CLA activity against intracellular pathogens. In conclusion, supramolecular assemblies combining cationic lipid and polymers, with or without traditional antibiotics, may find multiple possibilities of applications at pharmaceutical, medical, food and biotecnological fields.

Gold nanoparticles exhibit a combination of physical, chemical, optical, and electronic properties unique from all other nanotechnologies. These structures can provide a highly multifunctional platform with which to diagnose and treat diseases and can dramatically enhance a variety of photonic and electronic processes and devices. The work herein highlights some newly emerging applications of these phenomena as they relate to the targeted diagnosis and treatment of cancer, improved charge carrier generation in photovoltaic device materials, and strategies for enhanced spectrochemical analysis and detection. Chapter 1 introduces the reader to the design, synthesis, and molecular functionalization of gold nanotechnologies, and provides a framework from which to discuss the unique photophysical properties and applications of these nanoscale materials and their physiological interactions in Chapter 2. Chapter 3 discusses ongoing preclinical research in our lab investigating the use of near-infrared absorbing gold nanorods as photothermal contrast agents for laser ablation therapy of solid tumors. In Chapter 4, we present recent work developing a novel strategy for the targeted treatment of hormone-dependent breast and prostate tumors using multivalent gold nanoparticles that function as highly selective and potent endocrine receptor antagonist chemotherapeutics. In Chapter 5, we discuss a newly-emerging tumor-targeting strategy for nanoscale drug carriers which relies on their selective delivery to immune cells that exhibit high accumulation and infiltration into breast and brain tumors. Using this platform, we further investigate the interactions of nanoscale drug carriers and imaging agents to a transmembrane protein considered to be the single most prevalent and single most important contributor to drug resistance and the failure of chemotherapy. Chapter 6 presents work from a series of studies exploring enhanced charge carrier generation and relaxation in a hybrid electronic system exhibiting resonant interactions between photovoltaic device materials and plasmonic gold nanoparticles. Chapter 7 concludes by presenting studies investigating the contributions from so-called “dark” plasmon modes to the spectrochemical diagnostic method known as surface enhanced Raman scattering.

碩士
臺灣大學
醫學工程學研究所
98
To realize gene therapy and chemotherapy in multi-drug-resistant cancer cell, we develop carriers which can co-delivery gene and chemotherapy drug. The mPEG-PCL-PEI (M510i) tri-block polymers were synthesized by use mMPEG-PCL copolymer modified to PEI. The characteristic of these tri-block polymers were evaluated by 1H nuclear magnetic resonance and gel permeation chromatography. The critical micelle concentration (CMC) of micelle was evaluated by using pyrene as fluorescence probe. The particle size, zeta potential, and morphology of micelle was studied by dynamic light scattering and transmission electron microscopy. The results indicate that the paclitaxel loaded micelles and DNA complexes with micelles were 226 nm and 238 nm. The gene transfection efficiency was evaluated by used flow cytometry to evaluate green fluorescence protein (GFP) expression. The gene transfection efficiency performed better than PEI 25K in MCF-7 ADR cell. In siRNA experiments, we can transfect MDR-1 siRNA to silence P-glycoprotein expression 50%. In viro cytotoxicity, dual agent micelle of were tested of MCF-7 wt and MCF-7 ADR by MTT assy. These results suggested the PEG-PCL-PEI tri-block polymer as potential carriers for gene therapy and chemotherapy.

Multidrug resistance is a major problems associated with cancer chemotherapy. Efflux transports is one of the numerous mechanisms involved in multidrug resistance. P-glycoprotein is a transmembrane protein, responsible for drug efflux, which decreases drugs intracellular bioavailability, consequently decreasing their efficacy against cancer. Cancer growth and dissemination depends on the expression of transcriptional factors such as, Twist. Among other features, this protein is related with cells chemoresistance possible by regulation of multidrug resistance pathways including the P-glycoprotein expression. The herein study proposes to demonstrate if paclitaxel entrapped nanoparticles is an effective system in evading multidrug resistance mechanisms and if functionalization of a specific antibody against cancer stem cells receptors (anti-CD44v6) has the capability to target selectively these cells increasing nanoparticles efficacy. Therefore solid lipid nanoparticles were prepared and a breast cancer cell line (MDA-MB-436) was exposed to them in order to assess unloaded nanoparticles cytotoxic effects, increased pharmacologic efficacy of loaded nanoparticles relative to the free drug and their ability to evade multidrug resistance. The proposed solid lipid nanoparticles system proved to be capable of efficiently evading multidrug resistance mechanisms; however no improvement was added when these nanoparticles were functionalized with the antibody in the in vitro studies. However, the nanoparticles system is effective against multidrug resistance mechanisms.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
A resistência bacteriana apresenta-se nos dias de hoje como uma ameaça constante, com impacto não só ao nível da saúde da comunidade global como também na economia dos países. A ineficácia das quinolonas apresenta especial importância, uma vez que esta classe de antibióticos ocupa um lugar de destaque constituindo uma escolha comum de antibiotrapia.Com este trabalho pretende-se sistematizar os melhores métodos de produção, com vista à obtenção de características físico-químicas otimizadas para produção de formulações eficazes na erradicação de resistências. Estes novos sistemas de administração de fármacos minimizam os efeitos de bombas de efluxo, a formação de biofilmes, tendo ainda a capacidade de ultrapassar diversos problemas de farmacocinética. Para alcançar esse compromisso, foi realizada uma revisão sistemática da literatura para entender os principais usos das nanopartículas no cenário de resistência a antibióticos, bem como a integração e interpretação de uma série dados provenientes de experiências realizadas na Universidade de Coimbra, validando as informações descritas na literatura.Existem inúmeras classes de nanopartículas e optamos por abordar nanopartículas lipídicas, poliméricas e inorgânicas neste artigo devido à sua aplicação no contexto de resistências a antibióticos.Entre os processos de produção de nanopartículas lipídicas sólidas (SLN), a técnica de microemulsão apresentou os melhores tamanhos de partículas e ainda taxas de encapsulação mais eficientes. Quanto à produção de transportadores lipídicos nanoestruturados (NLC), o processo com o melhor tamanho de partícula foi a Homogeneização a Alta Pressão (HPH). Em relação às partículas poliméricas, as técnicas destacadas foram dessolvatação, coacervação e gelificação iónica. Os usos previstos de diferentes tipos de partículas nos respectivos contextos clínicos são discutidos em detalhes neste trabalho, considerando as diferenças nas propriedades farmacocinéticas e farmacodinâmicas.
The antibiotic resistance is a global threat to world’s public health, encompassing several challenges to human health as well as a huge economic burden. The unmet aimed efficiency of quinolones retains crucial interest and relevance since they are a common choice in antibiotherapy. This work aims to review the several nanoparticle production processes, in order to obtain delivery systems able to surpass bacteria’s widespread resistance. These new delivery systems minimize the efflux pump effects, the formation of biofilms and have the capacity of overcome pharmacokinetic issues. To achieve such commitment a critical analysis of literature was performed to understand the main usages of nanoparticles in antibiotic resistance setting, as well as a series of experiments performed in the University of Coimbra to validate whenever possible the information described by literature. Among numerous classes of nanoparticles, we have chosen to address lipid, polymeric and inorganic nanoparticles due to its appliance in the context of antibiotic resistances. Among production processes for Solid Lipid Nanoparticles (SLN), the microemulsion technique presented the best particle size and the most efficient encapsulation rate. As for the production of Nanostructured Lipid Carriers (NLC), the process with the best particle size was High Pressure Homogenization (HPH). Regarding polymeric particles, the highlighted techniques were desolvation, coacervation, and ionic gelation. The foreseen usages of different types of particles in the respective clinical settings are discussed in detail in this work, namely comprising the differences in pharmacokinetic and pharmacodynamic properties.

I. Gallium-hemoglobin Coated Silver Nanoparticles for Antimicrobial Photodynamic Therapy Against Bacterial Pathogens One of the mechanisms for bacterial pathogens’ hemin acquisition is through cell-surface hemin receptors (CSHRs), which are responsible for rapid hemin recognition. GaPpIX, as a hemin analog, can be rapidly taken up by CSHR-expressing bacteria, such as Staphylococcus aureus (S.aureus). Previous works shown that GaPpIX has aPDI activity at micromolar level of concentration following 10 seconds of 405-nm light exposure using LED array. The photosensitizing ability of GaPpIX can be further enhanced by incorporating with hemoglobin (GaHb) and 10 nm silver nanoparticles (AgNP). The results suggested a higher aPDI activity of GaHb-AgNP than any of its components against MRSA strains and neglectable cytotoxicity against keratinocytes. GaHb-AgNPs were also found having aPDI activity against intracellular MRSA and Mycobacterium abscessus but not effective against S. aureus biofilm. GaHb-AgNPs have no significant toxicity toward macrophages with concentrations lower than 22.64 μg/mL.

II. Synthesis and Properties of Magnetic Gold Nanoparticles

Superparamagnetic gold nanoparticles support hybrid magnetic and plasmonic properties that can be exploited for a variety of applications. In this paper we present new insights on the synthesis of magnetic gold nanoparticles (MGNPs) with an emphasis on efficiency, scalability, and waste reduction, supported by a comprehensive analysis of their physical and materials properties. Aqueous suspensions of colloidal Fe₃O₄ are conditioned with 5-kDa polyethylene glycol and L-histidine to mediate the nucleation and growth of gold by a mild reducing agent. Isotropic MGNPs on the order of 100 nm can be synthesized using scalable reaction conditions with Au:Fe mole ratios as low as 1:2 and cleansed with generally regarded as safe (GRAS) chemicals for the removal of residual iron oxide. High-resolution energy-dispersive x-ray imaging of individual MGNCs revealed these to be ultrafine composites of gold and SPIO rather than core–shell structures. The attenuated total reflectance infrared (ATR-IR) spectroscopy and Raman spectroscopy indicated that the cleansing step does change the optical properties of the synthesized MGNPs. Magnetometry of MGNCs in bulk powder form confirmed their superparamagnetic nature, with bulk moments between 6 to 7 emu/g.

博士
國立中興大學
化學工程學系所
105
In recent years, nanotechnology has been used for the development of drug delivery system for cancer therapy. Those nanocarriers can effectively accumulate in the tumor site via the enhanced permeability and retention (EPR) effect. However, multidrug resistance (MDR) is frequently encountered in patients undergoing long-term chemotherapy, leading to the failure of chemotherapy. One of the well-known mechanisms of MDR is drug efflux mediated by permeability glycoprotein (P-gp). The MDR cancer cells overexpress the P-gp on their cell membrane and efflux the drugs released from the nanocarriers. In this study, two drug delivery nanoparticle platforms were developed to depress the MDR cancer cells. In the first research, a pH-responsive cholesterol-PEG-coated solid lipid nanoparticle (C-PEG-SLN) carrying doxorubicin (DOX) capable of overcoming MDR breast cancer cells was developed in this work. The drug release behavior of the SLNs was studied at pH characteristic of the blood circulation (pH 7.4), the acidic tumor microenvironment (pH 6.0), and the subcellular acidic organelles (4.7) with succinate and PBS buffers, respectively. This result imply that the SLNs displayed the pH targeting behavior for the MDR cancer cells. In addition, the C-PEG (adducts) released from SLNs in endosome was transported to the cell membrane via endocytic recycling compartment pathway, which could limit cell membrane fluidity and thus inhibit the activity of P-gp also facilitated the therapeutic efficiency for the MDR cells. In the animal experiments, the growth of MCF-7/MDR xenografted tumor on nude mice treated with C-PEG-SLNs was inhibited to ca 400 mm3 in volume as compared with the PBS treatment group, 1140 mm3. No significant change in body weight of nude mice and the histology of organs and tumor after DOX-loaded SLNs administration show that the SLNs have no observable side effects. In the second research, a biocompatible graft copolymer, poly(γ-glutamic acid)-g-poly(lactic-co-glycolic acid) (γ-PGA-g-PLGA), was used as a major material to prepare the polymeric nanoparticles (DI-NPs) which is capable of simultaneously carrying doxorubicin (DOX) and indocyanine green (ICG) for the combinational therapy against MDR cancer cells. The DI-NPs exhibited the high payload loading capacity and superior stability in DMEM for 24 h. After the 808 nm laser irradiation, the DI-NPs displayed a pronounced cytotoxicity by photoactivated hyperthermia against MDR cancer cells. The increasing temperature also enhanced the DOX (base form) release from DI-NPs in MCF-7/MDR cancer cells. The CP2k-DI-NPs exhibited the strongest synergistic effect (CI = 0.46 < 1) for MDR cancer therapy. The more dual drugs accumulated in the tumor sites, the more therapeutic efficacy was approached for anti-MDR cancer treatment. In the in vivo results, the growth of MCF-7/MDR tumor on nude mice treated with CP2k-DI-NPs was inhibited to ca 120 mm3 in volume as compared with the PBS treatment group, 520 mm3. Analysis of the body weight of nude mice and the histology of organs and tumor receiving DI-NPs administration showed that the DI-NPs did not show side effects. Above all, we successfully used high biocompatible materials to develop two drug delivery system. Both of them showed superior therapeutic efficacy for MDR cancer cells. This indicates that the C-PEG-SLNs and CP2k-DI-NPs are promising platforms for the delivery of therapeutic agents for MDR cancer therapy.

碩士
中原大學
奈米科技碩士學位學程
97
Cancer multidrug resistance is associated with plasma membrane expression of P-glycoprotein which results to a reduction of the intracellular drug concentration after activation of P-glycoprotein efflux. In this study, an AB2 type amphiphilic block copolymer was synthesized from biodegradable methoxy poly (ethylene glycol) (mPEG) and poly (ε-caprolactone) (PCL) to encapsulate the antitumor drug, Doxorubicin (DOX). The initiator mPEG was first esterified with 2,2-bis (hydroxymethyl) butyric acid. Removal of the protective group by acidic resin afforded a copolymer with two hydroxyl chain terminals which was subsequently used in a ring-opening polymerization of ε-caprolactone. The AB2 copolymer mPEG-(PCL)2 was characterized by Fourier transform infrared spectroscopy and 1H nuclear magnetic resonance spectroscopy. By increasing the 2,2-bis (hydroxymethyl) butyric acid content in the copolymer, the melting point can decrease from 62.29 oC to 60.73 oC. The molecular weight of the copolymer was calculated as 15,245 g/mol by gel permeation chromatography and 19,312 g/mol by 1H nuclear magnetic resonance. The amphiphilic block copolymer self-assembled into micelles and the critical micelle concentration was 43.7×10-3 mg/mL. The particle size of the empty micelle was measured by dynamic light scattering and found to be 95.1 nm. After loading with doxorubicin, the particle size was 21.4 nm. The drug encapsulation was measured at 22.29% by UV-vis spectrophotometer. The in vitro release study indicated that 50% of the drug was released from micelles at pH 5 acetate buffer and 40 % at pH 7.4 phosphate buffer in the duration of 48 hours. Incubation of macrophage cells with the empty micelles resulted in 0.420 O.D./mg protein and 0.413-0.472 O.D./mg protein NO production for the control and micelle, respectively. This indicated that the micelles could avoid recognition by macrophage cells. Rhodamine 123 assay by flow cytometry and western blot were used to monitor the relative P-glycoprotein expression in human breast cancer cell lines MCF-7/WT and MCF-7/ADR. The results showed that the fluorescence of rhodamine 123 was stronger in MCF-7/WT than MCF-7/ADR. Western blot analysis detected a single band for P-glycoprotein at 170 kDa. The IC50 value of drug-loaded micelle for MCF-7/WT and MCF-7/ADR were 0.285 μg/mL and 7.476 μg/mL, respectively. These values are 7.9-fold higher than the IC50 of free drug for MCF-7/WT and MCF-7/ADR at 0.036 μg/mL and 0.937 μg/mL, respectively. This can be due to the slow release rate of DOX from the micelles. The cell uptake study by confocal laser scanning microscopy (CLSM) showed that drug-loaded micelles accumulated mostly in the cytoplasm instead of the nuclei through endocytosis. In contrast, free drug diffused throughout the cell. In addition the number of cell uptake under the same fluorescence intensity was measured to be 83.16 % and 50.91 % for drug-loaded micelles and free drug in MCF-7/ADR cells. In conclusion, the AB2 copolymer was able to overcome multidrug resistance of breast cancer cells as it can accumulate more in MCF-7/ADR cells compared to free drug. Future in vivo studies could focus on how the drug delivery system can inhibit the tumor growth and prolong the survival rate.