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The development of techniques for colloidal nanoparticle synthesis has allowed scientists to fabricate materials that can manipulate light on a scale that is small even compared to the wavelength of the light itself. This ability has led to the development of myriad and diverse applications of nanostructures in wide-ranging fields. This thesis focuses on the investigation and exploitation of nanoscale material properties in the fields of medicine and energy. The unique optical properties of nanoparticles arise from their size and their high surface area to volume ratios compared to bulk materials. As a result of this relationship, the surface characteristics of nanoparticles generally dominate their properties, whereas in bulk materials the surface atoms have very little bearing on the properties of the composite. Chapter 1 gives an introduction to nanoparticles and their optical properties, including a discussion of the plasmon resonance and the properties imbued upon nanoparticles possesing such a resonance as well as the applicability of these properties that will be explored in the subsequent chapters. Chapter 2 presents a study of the interaction of cationic, hydrophobic gold nanoparticles as probes to elucidate specific regions of interest on cell surfaces. The high imaging contrast of gold nanoparticles in electron microscopy allows for visual, macroscopic observation of the aggregation patterns formed by these nanoparticles on cell surfaces. Plasmon resonant coupling between proximal nanoparticles is exploited in order to monitor nanoprobe binding and localization over time with the use of extinction spectroscopy. The role of surface proteins in the nanoparticle-cell surface interaction is elucidated, generating composite data with relevance in pharmaceutical development and pharmacokinetics. Additionally, bacteria strain-dependent toxicity is observed and subsequently investigated for smaller gold nanoparticle probes, demonstrating a potential use for nanoparticles as strain-specific antibiotics. The development of affordable, effective antibiotic technology is one of the major scientific challenges of our time; infections from pathogen-infested drinking water alone account for millions of deaths each year worldwide. In Chapter 3, we investigate the use of titanium dioxide as an inexpensive method to harness solar energy to split water into reactive species and thereby decontamitate solutions of E. coli. Though titanium dioxide is an excellent catalyst for water splitting, it requires UV irradiation, which is fairly lacking in the solar emission spectrum. Further, recuperation of titanium dioxide nanoparticles from solution is non-trivial, and its immobilization into a film greatly limits its surface area and charge carrier efficiency, thereby limiting its activity. We treat both the poor visible light absorption capability as well as the surface area limitation in this study. CdS semiconductor nanocrystals are used to extend the absorption edge of TiO₂ further into the visible light region of the spectrum by providing for lower-energy photon absorption and charge injection into titanium dioxide. TiO₂ is also electrochemically anodized to generate TiO₂ nanotube arrays, which have greatly increased surface area as well as more efficient charge transfer properties compared to thin films of TiO₂ nanoparticles. The utility of nanoparticles in increasing the light absorption of other systems continues as a theme in the work presented in the next two chapters. Chapter 4 ex- amines the plasmonic enhancement of the solar energy conversion in a biomimetic system. In this endeavor, we enhance the photocurrent generated by a light-transducing, proton-pumping protein, bacteriorhodopsin, in a 3-dimensional wet electrochemical cell. First, we increase the overall charge carrier separation with the use of a proton- selective membrane in order to minimize ionic depolarization in the cell. We then use plasmonic nanoparticles to exploit an irregularity in the bacteriorhodopsin photocycle known as the blue light effect. This effect shortens the timescale of the photocyle by more than 99% via blue photon absorption, but it has a very low natural occurrence. Plasmonic nanoparticles tuned to the blue wavelength region increase the flux of blue photons on a local level and thereby increase the overall photocurrent generation. We first examine the importance of nanoparticle field strength to photocurrent enhancement using silver nanospheres with different capping shell thicknesses. We then consider the trade-off between (1) using a nanoparticle with a plasmon resonance tuned perfectly to the blue wavelength region and (2) using a nanoparticle with a stronger field intensity but weaker energetic presence in the blue. By minimizing ionic depolarization, minimizing shielding of the plasmon electromagnetic field, and maximizing the field strength while maintaining the plasmon frequency at the proper wavelength, we demonstrate an enhancement of 5,000-fold in the photocurrent production by bacteriorhodopsin. Chapter 5 explores a variation on the theme of Chapter 4 with an application in cancer therapeutics. Here, a photodynamic cancer drug, protoporphyrin IX (PpIX), is incorporated into complexes with silver nanospheres, gold nanospheres, and gold nanorods. Each of these nanoparticles displays a plasmon resonance in a different region of the spectrum, with consequent different overlap with the absorption or emission of the drug. Photodynamic therapeutic potential is measured in situ and in vivo, and the drug activity is shown to be strongest when drug absorption overlaps with plasmon resonance. Absorption by electronic excitations in the particle crystal lattice is shown to function as a competitive light filter and decrease drug activity. Additionally, the method of attachment of the drug to the nanoparticle is examined. Maximum enhancement of drug activity is shown to require the drug to remain bound close to the nanoparticle surface, where the electromagnetic field strength is highest. This plasmonic enhancement effect on drug activity is shown to outstrip the increase in drug activity seen when using the nanoparticle solely as a delivery platform. In Chapter 6, some synthetic techniques are presented for various nanomaterials. Included are syntheses for gold, silver, and semiconductor nanoparticles of a variety of shapes and sizes as well as for TiO₂ nanotube arrays. The relationship of the ratio of capping agent to metal salt is explored for gold nanospheres, and a method for facile tuning of the longitudinal plasmon resonance displayed by gold nanorods is presented. Synthetic techniques are also presented for the nanoparticles whose applications are explored in the preceding chapters.

Cyanine dyes that absorb light in the near infrared to far red region of the electromagnetic spectrum are desirable as photosensitizers for photodynamic cancer therapy. Light of wavelengths in this range is able to deeply penetrate tissue allowing for practical in vivo use of these dyes. A series of three structurally similar pentamethine cyanine dyes that absorb light ~800 nm to ~500 nm was tested to determine the effects of structural influences on the yields of supercoiled DNA photo-converted to nicked or linear forms. Possible mechanisms and optimal parameters for near- quantitative DNA photocleavage with a symmetrical quinoline pentamethine cyanine dye are discussed.

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department: Department of Biophysics and Physical Chemistry Candidate: Pavel Cermak Supervisor: Assoc. Prof. Veronika Novakova, PhD. Title of Thesis: Preparation and photophysical evaluation of tetra-3,4- pyridoporphyrazines suitable for the photodynamic therapy Tetra-3,4-pyridoporphyrazines (TPyPz) are aza-analogues of phthalocyanines. Their large system of conjugated bonds enables them to absorb light in the red part of the absorption spectrum. Due to their ability to produce singlet oxygen, they can be potentially used as photosensitizers in photodynamic therapy (PDT). Its mechanism is based on co-functioning of three elements - photosensitizer, light and oxygen. Photosensitizer excited by light absorption transfers its energy into tissue oxygen, thus, creating cytotoxic singlet oxygen. This method is beneficial for its high selectivity, low toxicity, minimal invasion and fast effect. The aim of this work was to synthetize and study water-soluble TPyPz suitable for PDT. Water solubility was achieved by quarternized amines, forming of salts or using suitable delivery systems (hydrophilic emulsion). Hydrophilicity was also increased by introduction of hydrophilic non-charged substituents (OH). At first, appropriate precursors for...

Master of Science / Department of Anatomy & Physiology / Deryl L. Troyer / Breast cancer ranks second as a cause of cancer death in women in the USA. Detection of early tumors and tumor-targeted treatments could decrease the problems associated with breast cancer management. Photodynamic therapy (PDT) is a cancer treatment that uses a photosensitizer and a specific wavelength of light and is currently in clinical trials for breast cancer. When tumor cells which have absorbed photosensitizer are exposed to the correct wavelength of light, reactive oxygen species are generated, resulting in tumor cell death. Poor tissue penetration of light is a major limitation in PDT, restricting its use to treatment of localized tumors. Light generation at the tumor area might increase the effectiveness of PDT. Polymorphonuclear neutrophils (PMNs) are known to often infiltrate breast adenocarcinoma, and their activatation in tumor stroma produces luminescence in the presence of luminol. Here, we hypothesized that luminol can be used as a theranostic agent for luminescence-based early tumor detection (diagnosis) and in situ PDT (treatment). BALB/c mice were transplanted with 4T1 mammary adenocarcinoma cells to establish a breast adenocarcinoma model. The early tumor detection objective was tested by daily intraperitoneal injection of luminol and in vivo luminescence imaging. To test the PDT treatment objective,the photosensitizer 5-aminolevulinic acid (ALA) and luminol were administered to mice through intraperitoneal and intravenous routes, respectively. This treatment regimen was repeated six times and ALA alone/luminol alone/saline treated tumor-bearing mice were used as controls. Results demonstrated that luminol allowed detection of activated PMNs only two days after 4T1 cell transplantation, even though tumors were not yet palpable. Relative differences in the increase of tumor volume and final tumor weights were analyzed to test the in situ PDT. Analysis of the data showed luminol treatments resulted in breast adenocarcinoma tumor growth attenuation. In conclusion this study provides evidence that luminol can be a theranostic agent for breast adenocarcinoma.

La thérapie photodynamique est fondée sur l’activation sélective par la lumière de médicaments appelés photosensibilisateurs. Non toxique en l’absence d’excitation lumineuse, le photosensibilisateur est capable, une fois excité, de transférer son énergie pour former de l’oxygène singulet qui induit la mort de la cellule. L’utilisation d’une excitation biphotonique offre de nouvelles perspectives pour la thérapie photodynamique du cancer. En effet, la dépendance quadratique de l’absorption à deux photons (ADP) avec l’intensité du laser permet une très grande sélectivité spatiale, ce qui rend possible un traitement plus sélectif des tumeurs, et la lumière infra-rouge utilisée permet le traitement de tumeurs plus profondes. Dans ce travail, nous avons développé une famille de nano-objets tout organiques pour la thérapie photodynamique biphotonique. Ils présentent des sections efficaces d’ADP très élevées dans la gamme spectrale d’intérêt biologique et leur efficacité a été démontrée in vitro. / Photodynamic therapy is based on the selective activation by light of drugs called photosensitizers. Non-toxic in the absence of excitation light, the photosensitizer is able, upon excitation, to transfer energy to produce singlet oxygen, which induces cell death. Two-photon excitation offers new perspectives for photodynamic therapy of cancer. Indeed, the quadratic dependence of two-photon absorption (TPA) with the laser intensity allows high spatial selectivity, which enables a more selective treatment of tumors, and the use of infrared light allows treatment of deeper tumors. In this work, we have developed a family of fully organic nano-objects for two-photon photodynamic therapy. They exhibit very high TPA cross-sections in the biological spectral range of interest and their efficiency has been demonstrated in vitro.

Objetivo: Avaliar, in vivo, o efeito do tratamento endodôntico em sessão única utilizando a Terapia Fotodinâmica Antimicrobiana (aPDT) no reparo de lesões periapicais induzidas em dentes de cães, por meio da avaliação histopatológica e imunohistoquímica da angiogênese e de marcadores de formação óssea. O tratamento endodôntico em duas sessões com curativo de demora à base de hidróxido de cálcio (CH) foi utilizado como controle. Métodos: Lesões periapicais foram induzidas em 48 pré-molares superiores e inferiores de 6 cães, com 12 meses de idade. Após instrumentação dos canais radiculares, os dentes foram divididos, aleatoriamente, em 4 grupos: CH/120dias (d) e CH/180dias (d): canais radiculares preenchidos com curativo à base de CH; aPDT/120d e aPDT/180d: canais radiculares condicionados com fotossensibilizador à base de fenotiazina (10 mg/mL), por 1 minuto e irradiados com laser de diodo em toda a extensão dos canais, conforme as recomendações do fabricante. Em seguida, todos os canais radiculares foram obturados com cimento AH Plus e, após 120 ou 180 dias, os animais foram eutanasiados e os blocos contendo dentes e tecido ósseo foram submetidos ao processamento histotécnico e à coloração de hematoxilina e eosina (HE) para a análise descritiva da região periapical e mensuração das lesões periapicais, em microscopia convencional, e contagem de vasos sanguíneos sob luz convencional e no modo fluorescente. A análise imunohistoquímica foi realizada para avaliação dos marcadores da formação óssea osteopontina (OPN) e fosfatase alcalina (ALP). Os dados obtidos foram analisados estatisticamente utilizando os testes two-way ANOVA e qui-quadrado, com nível de significância de 5%. Resultados: Aos 120 dias, os dentes do grupo CH/120d apresentaram processo de reparo avançado, com ligamento periodontal apenas ligeiramente aumentado, presença abundante de fibras colágenas e escassas células inflamatórias. Os dentes do grupo aPDT/120d apresentaram o ligamento periodontal moderadamente aumentado e o infiltrado inflamatório era moderado. Poucas fibras colágenas foram observadas. Aos 180 dias, o mesmo padrão foi observado. As lesões periapicais nos grupos tratados com curativo à base de hidróxido de cálcio foram menores que as lesões nos grupos tratados com aPDT (p<0,001), e apresentaram maior número de vasos sanguíneos (p<0,0001), independentemente dos períodos de avaliação. Além disso, os dentes dos grupos tratados com pasta à base de hidróxido de cálcio apresentaram imunomarcação significativamente mais intensa para ALP e OPN (p<0,001), em ambos os períodos. Conclusões: Embora o tratamento com a aPDT tenha estimulado a angiogênese e a expressão dos marcadores da formação óssea, o tratamento endodôntico realizado em duas sessões empregando curativo à base de hidróxido de cálcio estimulou mais intensamente esses processos e promoveu melhor reparo das lesões periapicais / Aim: The aim of this study was to evaluate the in vivo effect of one-session endodontic treatment with antimicrobial photodynamic therapy (aPDT) in the repair of apical periodontitis, in dogs\' teeth, by histopathologic evaluation and imunohistochemistry for angiogenesis and bone formation markers. Two-session treatment with a calcium hydroxide (CH) dressing was used as control. Methods: Apical periodontitis were induced in 48 upper and lower premolars of six 12-monthold dogs. After root canals instrumentation, teeth were randomly divided into 4 groups: CH/120days (d) and CH/180days (d): root canals filled with CH-based dressing; aPDT/120d and aPDT/180d: root canals conditioned with phenothiazinebased photosensitizer (10 mg/mL) for 1 minute and irradiated with diode laser throughout the canals and according to the manufacturer\'s recommendations. Root canals were filled with AH Plus cement, and after 120 or 180 days, the animals were euthanized and teeth were submitted to histotechnical processing and HE staining for description of the periapical region and measurement of apical periodontitis in conventional microscopy and for counting blood vessels under conventional and fluorescent microscopy. Immunohistochemical analysis was performed to evaluate the bone formation markers osteopontin (OPN) and alkaline phosphatase (ALP). Data were statistically analyzed using two-way ANOVA and chi-square test with a significance level of 5%. Results: At 120 days, teeth in Group CH/120d presented the periodontal ligament only slightly enlarged with advanced repair and abundant collagen fibers. Inflammatory cells were scarce. Teeth in group aPDT/120d presented the periodontal ligament moderately enlarged and the inflammatory infiltrate was moderate. Few collagen fibers were observed. At 180 days, the same pattern was observed. Apical periodontitis in CH-treated groups were smaller than the lesions in aPDT-treated groups (p<0.001) and had a greater number of blood vessels (p <0.0001), regardless of evaluation periods. The teeth treated with calcium hydroxide showed significantly more intense immunostaining for ALP and OPN (p<0.001), in both periods. Conclusions: Although aPDT has stimulated angiogenesis and the expression of bone formation markers, the two-session endodontic treatment with a calcium hydroxide-based dressing stimulates them more intensely and promoted better apical periodontitis repair

O objetivo do presente estudo foi avaliar, in vitro, o efeito de diferentes tempos de pré-irradiação do fotossensibilizador na terapia fotodinâmica em biofilmes formados por Streptococcus mutans e Candida albicans, por meio da avaliação da carga microbiana. Os fatores em estudo foram: tempos de pré-irradiação do fotossensibilizador em 3 níveis (1, 2 ou 5 minutos). Para o controle do biofilme dentário cariogênico com aPDT foi utilizado o azul de metileno (0,01%) associado ao laser de diodo (&lambda;=660 nm). O digluconato de clorexidina (CHX a 0,12%) e a solução salina foram utilizados como controle positivo e negativo, respectivamente. O delineamento do estudo foi realizado em blocos completos e casualizados, sendo a amostra composta por 15 culturas de biofilmes de S. mutans, divididas aleatoriamente em 5 grupos e 15 culturas de C. albicans, também divididas em 5 grupos. O experimento foi realizado em triplicata (n=3) e as variáveis de resposta foram obtidas por meio de análise quantitativa da viabilidade bacteriana, expressa em unidades formadoras de colônia (UFC) por mm2 da área do espécime. Os dados obtidos foram analisados com o auxílio do teste one-way ANOVA e pós-teste de Tukey. Todas as análises foram efetuadas por meio do programa Graph Pad Prism 4.0, com nível de significância de 5%. Para o grupo de S. mutans, apenas a solução salina apresentou diferença estatisticamente significante quando comparada aos demais tratamentos (p<0.05), ou seja, o tratamento com aPDT, independentemente do tempo de irradiação aplicado, foi semelhante ao tratamento com CHX e ambos foram mais eficazes na redução do biofilme cariogênico, em comparação à solução salina. Para o grupo de C. albicans não houve diferença estatística entre os grupos (p>0.05). Portanto, pode-se concluir que o tratamento com aPDT diminuiu o número de UFCs de S. Mutans de forma semelhante à CHX, independentemente do tempo de pré-irradiação aplicado. Não foi possível constatar nenhum efeito desta terapia e dos diferentes tempos de pré-irradiação sobre o biofilme de C. albicans. Desta forma, o tempo de pré-irradiação de 1 minuto pode ser utilizado com o objetivo de reduzir a carga microbiana de S. Mutans. / The aim of the present study was to evaluate, in vitro, the effect of different pre-irradiation times of the photosensitizer in photodynamic therapy in biofilms formed by on by Streptococcus mutans and Candida albicans, through the evaluation of the microbial load. The factors under study were: times of pre-irradiation of the photosensitizer in 3 levels (1, 2 or 5 minutes). For the control of the cariogenic dental biofilm with aPDT, methylene blue (0.01%) was used in association with the diode laser (&lambda;=660 nm). Chlorhexidine digluconate (0.12% CHX) and saline were used as positive and negative controls, respectively. The study design was carried out in complete and randomized blocks. The sample consisted of 15 S. mutans biofilms cultures, randomly divided into 5 groups and 15 C. albicans cultures, also divided into 5 groups. The experiment was performed in triplicate (n = 3) and the response variables were obtained through quantitative analysis of bacterial viability, expressed in colony forming units (CFU) per mm2 of the specimen area. The data were analyzed with the aid of the ANOVA one-way test and Tukey\'s post-test. All analyzes were performed using the Graph Pad Prism 4.0 program, with a significance level of 5%. For the S. mutans group, only the saline solution presented a statistically significant difference when compared to the other treatments (p <0.05), that is, the treatment with aPDT, irrespective of the irradiation time applied, was similar to the treatment with CHX and both were more effective in reducing cariogenic biofilm compared to saline. For the group of C. albicans there was no statistical difference between the groups (p> 0.05). Therefore, it can be concluded that the treatment with aPDT reduced the number of CFUs of S. Mutans in a similar way to CHX, independently of the pre-irradiation time applied. No effect of this therapy or of the different pre-irradiation times on the C. albicans biofilm could be observed. In this way, the pre-irradiation time of 1 minute can be used to reduce the microbial load of S. mutans.

<p>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 <i>Staphylococcus aureus</i> (<i>S.aureus)</i>. 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 <i>Mycobacterium abscessus </i>but not effective against <i>S. aureus</i> biofilm. GaHb-AgNPs have no significant toxicity toward macrophages with concentrations lower than 22.64 μg/mL.</p> <p> </p> <p>II. Synthesis and Properties of Magnetic Gold Nanoparticles</p> <p> 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.</p>

Photodynamic therapy (PDT) is a treatment modality for cancer. It combines selective accumulation of chemical compounds, called photosensitizers (PS), with light to irreversibly damage cancer cells via oxidative stress. The main goal of this thesis was to study photosensitizers represented by a unique group of newly synthesized porphyrin derivatives with glycol chain substitution. Glycol-functionalized porphyrins containing one to four low molecular weight glycol chains that are linked via ether bonds to the meta-phenyl positions of meso-tetraphenylporphyrin (mTPP(EG)1-4) were compared with fluorinated (pTPPF(EG)4) and nonfluorinated (TPP(EG)4) derivatives having glycol chains in para-phenyl positions. The cellular uptake and photodynamic activity was significantly dependent on terminal groups of the glycol substituent. Hydroxy glycol porphyrins, in contrast with methoxy glycol porphyrins, exhibited efficient intracellular transport and high induction of apoptosis in tumor cell lines in vitro. After initial testing effective prototype hydroxy ethylene glycol derivatives were selected and analyzed in detail. Para derivatives pTPP(EG)4 and pTPPF(EG)4 accumulated mainly in lysosomes whereas meta derivatives mTPP(EG)1-4 in the endoplasmic reticulum (ER). Position of ethylene glycol chain on the...