This study evaluated the ability of photodynamic therapy (PDT), chitosan nanoparticles (CSnp), or their combination, to inhibit bacterial collagenase-mediated degradation of collagen. Rat type 1 fibrillar collagen matrices were untreated or treated with 2.5% glutaraldehyde (GD), 2.5% GD followed by 1% CSnp, 1% CSnp, PDT, or 1% CSnp followed by PDT. Samples, except untreated controls, were exposed to Clostridium histolyticum collagenase. The soluble digestion products were assessed by hydroxyproline assay and the remaining adherent collagen was quantified by picrosirius red (PSR) staining. Collagen treated with CSnp, PDT, or a combination of CSnp and PDT, exhibited less degradation than controls. The abundance of posttreatment residual collagen correlated with the extent of degradation. Fourier transform infrared (FTIR) spectroscopy analysis showed that PDT treatment enhanced collagen cross-linking. Immunoblotting of sedimented CSnp indicated that CSnp and collagenase bound with low affinity. However, CSnp-bound collagenase showed a significant reduction in collagenolytic activity compared with controls.

This study evaluated the ability of photodynamic therapy (PDT), chitosan nanoparticles (CSnp), or their combination, to inhibit bacterial collagenase-mediated degradation of collagen. Rat type 1 fibrillar collagen matrices were untreated or treated with 2.5% glutaraldehyde (GD), 2.5% GD followed by 1% CSnp, 1% CSnp, PDT, or 1% CSnp followed by PDT. Samples, except untreated controls, were exposed to Clostridium histolyticum collagenase. The soluble digestion products were assessed by hydroxyproline assay and the remaining adherent collagen was quantified by picrosirius red (PSR) staining. Collagen treated with CSnp, PDT, or a combination of CSnp and PDT, exhibited less degradation than controls. The abundance of posttreatment residual collagen correlated with the extent of degradation. Fourier transform infrared (FTIR) spectroscopy analysis showed that PDT treatment enhanced collagen cross-linking. Immunoblotting of sedimented CSnp indicated that CSnp and collagenase bound with low affinity. However, CSnp-bound collagenase showed a significant reduction in collagenolytic activity compared with controls.

No description available.

The motivation of this study lies in the necessity for a microfocal therapy to specifically target diseased areas in vascular pathologies such as age-related macular degeneration (AMD). AMD is the most common cause of legal blindness among people over the age of 60 in developed countries. This degenerative condition affects the macula, the central region of the retina, severely impairing detailed vision and hindering everyday activities. Worldwide, 25-30 million people live with some form of AMD. Among them, ~10% suffer from the more advanced and damaging form, wet-AMD, which causes rapid and severe loss of central vision. To date, there is no cure or long-term alternative for this degenerative disease despite intensive research efforts. With recent developments in biophysical tools and experimental procedures, in this study, we demonstrate a highly-localized therapeutic option: two-photon (2-photon) photodynamic therapy (PDT) that could be advantageous for the cure of wet-AMD, either alone or in combination with recently discovered anti-angiogenic therapies. This new approach offers selective targeting of the diseased area, thus minimizing damage to the surrounding sensitive healthy eye tissues, which is a major concern with the clinically-used, standard wide-beam, one-photon (1-photon) PDT. The objective of the research was to test the feasibility of microfocal 1-photon and the inherently localized 2-photon PDT, their optimization and also to evaluate the efficacy of existing 1-photon and novel 2-photon photosensitizers. In this thesis, I illustrated the in vitro (endothelial cell monolayer) and in vivo (window chamber mouse (WCM)) models that can be used to quantitatively compare the 2-photon efficiency of photosensitizers. Using the in vitro model, I compared the 2-photon efficacy of clinically used 1-photon PDT drugs Photofrin and Visudyne, and showed that the Visudyne is an order of magnitude better 2-photon photosensitizer than Photofrin. With the WCM model, I demonstrated a novel designer 2-photon photosensitizer is 20 times more efficient than Visudyne for single vessel occlusion. I also generated the drug and light dose reciprocity curve for localized single-vessel microfocal PDT. This is a necessary step towards applying the method to the relevant ocular models of AMD, which is the next phase for this research.

The anticancer properties of dirhodium tetraacetate were discovered in the 1970's, and subsequently motivated the research of several dirhodium paddlewheel derivatives. The promising results of this research led the Dunbar group to investigate the biological properties of dirhodium partial paddlewheel compounds. Previous work in our group has focused on dirhodium carboxylate derivatives with a series of diimine ligands, namely 1,10-phenanthroline (phen), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq), dipyrido[3,2a:2',3'c] phenazine (dppz), and benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) (dppn). Current research has expanded this diimine series by substituting the carobxylate bridging group with p-methoxyphenylphosphine (PMP). This new series of compounds was characterized by several techniques, including: X-Ray crystallography, 1H NMR spectroscopy, and electronic absorption spectroscopy. The cytotoxicity of these compounds towards HeLa cells was investigated in presence and absence of light in an effort to investigate the ability to use these compounds as photodynamic therapy (PDT) agents. Cytotoxicity measurements were carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. It was found that in the dark [Rh2(PMP)2(dppz)2][BF4]2 (the dppz derivative of the dirhodium PMP compound) had no cytotoxicity towards HeLa cells, but experienced a 7 fold increase in cytotoxicity upon irradiation (with lambdai_rr equal to 350 nm). This dramatic increase in cytotoxicity upon irradiation makes this compound a potential PDT agent. Diiridium (II,II) compounds were prepared in a dual attempt to determine how the properties of the dirhodium core effect the biological activities of these compounds, as well as investigate the biological activity of a set of compounds that has yet to be explored. The compound [Ir2(DTolF)2(CH3CN)6][BF4]2 was chosen because it has a well understood dirhodium analogue, and it is a known compound. However, it was discovered that there was a potential silver contamination in the final product, stemming from the silver trifluoroacetate oxidant used during synthesis. Consequently, a new method of preparing this compound was required. The new synthetic pathway for the diiridium compound [Ir2(DTolF)2(CH3CN)6][BF4]2 was devised, and the cytotoxicity and photocytotoxicity studies were performed for the first time (to our knowledge) on a diiridium (II,II) compound. Despite the stability of the compound, it was determined to be highly toxic, both in the dark and upon irradiation.

Made available in DSpace on 2014-10-09T12:27:57Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:00:54Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:27:57Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:00:54Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Devant l’augmentation de l’incidence des cancers de prostate de faible risque, les thérapies focales apparaissent comme une alternative séduisante entre la surveillance active et les traitements radicaux classiques. Les thérapies Laser (Thérapie photodynamique [PDT] et thermothérapie laser interstitielle [LITT]) pourraient être des modalités d’application de cette thérapie focale.Dans la première partie de cette thèse, nous discutons du concept de thérapie focale, et de la place des thérapies Laser par rapport aux différentes autres modalités énergétiques. Le premier chapitre de cette partie est consacré à l’émergence de la thérapie focale, à ses bases histopathologiques et à l’importance de l’imagerie dans son application. L’échographie et l’imagerie par résonance magnétique (IRM) représentent des outils indispensables pour l’aide au diagnostic, la sélection des patients et le guidage du traitement focal. Les avantages et inconvénients ainsi que les principaux résultats cliniques des différentes modalités énergétiques utilisées pour la thérapie focale sont présentés dans le deuxième chapitre de cette partie. Nous dressons un état de l’art de la PDT et de la LITT pour le traitement focal dans un troisième chapitre. Ces thérapeutiques Laser sont actuellement en développement clinique. Ces deux modalités de traitement reposent sur des principes physiques différents, mais leur application à la thérapie focale pose des problématiques identiques (planification dosimétrique, guidage du traitement interstitiel). Nous précisons les développements actuellement en cours pour résoudre ces problématiques et notamment les travaux de notre unité de recherche. La deuxième partie de cette thèse est dédiée au développement expérimental de LITT pour le cancer de prostate menée au sein de notre unité de recherche. Dans le premier chapitre de cette partie, nous présentons la mise en place d’un modèle animal (Rat Copenhagen avec greffe hétérotopique d’un adénocarcinome prostatique syngénique Dunning R3327-AT2) adapté au traitement par LITT focale. Nous décrivons le monitoring de cette thérapie sous IRM et échographie et démontrons la reproductibilité des volumes de nécrose induits pour un niveau d’énergie donné. Le deuxième chapitre de cette partie est consacré à la mise en place d’un outils de planification dosimétrique adapté à notre modèle in vivo et basé sur la diffusion de chaleur au sein des tissus et au calcul des dommages cellulaires. Enfin dans le troisième chapitre, nous proposons la mise en place d’un fantôme de prostate anatomiquement valide permettant de simuler et d’expérimenter des procédures focales de LITT. Ce fantôme compatible avec un monitoring sous échographie et sous IRM pourrait également être utilisé pour le développement d’autres modalités énergétiques de thérapie focale. / Given the increased incidence of prostate cancer low-risk, focal therapies appear as an attractive alternative between active surveillance and radical treatments. Laser therapies (photodynamic therapy [PDT] and interstitial laser thermotherapy [LITT]) could be modalities of application of this focal therapy.In the first section of this thesis, we discuss the concept of focal therapy, and the place of laser therapies compared to several other energy modalities. The first chapter of this section is devoted to the emergence of focal therapy, its histopathologic bases and the importance of imaging in its application. Ultrasonography and magnetic resonance imaging (MRI) are essential tools to aid diagnosis, patient selection and guidance of focal treatment. The advantages and disadvantages and the main clinical outcomes of different energy modalities used for focal therapy are presented in the second chapter of this section. We summarize a state of the art of PDT and LITT for the focal treatment in a third chapter. These Laser therapies are currently in clinical development. Both treatment modalities are based on different physical principles but their application to focal therapy raises identical issues (treatment planning, guiding interstitial treatment). We specify the developments currently underway to address these issues and particularly the work of our research unit.The second section of this thesis is dedicated to the experimental development of LITT for prostate cancer conducted in our research unit. In the first chapter of this section, we present the development of an animal model (rat Copenhagen with heterotopic and syngenic prostatic adenocarcinoma transplant Dunning R3327 -AT2) for processing focal LITT. We describe the monitoring of this therapy under ultrasonography and MRI and demonstrate the reproducibility of the volumes of necrosis induced for one given energy level. The second chapter of this section is devoted to the establishment of a treatment planning tools adapted to our in vivo model and based on tissue heat diffusion and calculation of induced cell damages. Finally in the third chapter, we propose the establishment of an anatomically valid prostate phantom to simulate and test focal LITT procedures. This phantom compatible with ultrasonography and MRI monitoring could also be used for the development of other focal therapy energy modalities.

Recent progress in nanotechnology has attracted interest to a biomedical application of the carbon nanoparticle C60 fullerene (C60) due to its unique structure and versatile biological activity. In the current study the dual functionality of C60 as a photosensitizer and a drug nanocarrier was exploited to improve the efficiency of chemotherapeutic drugs towards human leukemic cells. Pristine C60 demonstrated time-dependent accumulation with predominant mitochondrial localization in leukemic cells. C60’s effects on leukemic cells irradiated with high power single chip LEDs of different wavelengths were assessed to find out the most effective photoexcitation conditions. A C60-based noncovalent nanosized system as a carrier for an optimized drug delivery to the cells was evaluated in accordance to its physicochemical properties and toxic effects. Finally, nanomolar amounts of C60-drug nanocomplexes in 1:1 and 2:1 molar ratios were explored to improve the efficiency of cell treatment, complementing it with photodynamic approach. A proposed treatment strategy was developed for C60 nanocomplexes with the common chemotherapeutic drug Doxorubicin, whose intracellular accumulation and localization, cytotoxicity and mechanism of action were investigated. The developed strategy was revealed to be transferable to an alternative potent anticancer drug – the herbal alkaloid Berberine. Hereafter, a strong synergy of treatments arising from the combination of C60-mediated drug delivery and C60 photoexcitation was revealed. Presented data indicate that a combination of chemo- and photodynamic treatments with C60-drug nanoformulations could provide a promising synergetic approach for cancer treatment. / Kürzliche Fortschritte in der Nanotechnologie haben Interesse an einer biomedizinischen Anwendung des Kohlenstoffnanopartikels C60 Fulleren (C60) aufgrund seiner einzigartigen Struktur und breiten biologischen Aktivität geweckt. In der aktuellen Studie wurde die doppelte Funktionalität von C60 als Photosensibilisator und als Wirkstoff-Nanoträger genutzt, um die Wirkung von Chemotherapeutika auf menschliche Leukämiezellen zu verbessern. C60 alleine zeigte in den Zellen eine zeitabhängige Akkumulation mit vorherrschender mitochondrialer Lokalisation. Die Wirkung von C60 auf Leukämiezellen, die mit unterschiedlicher Wellenlänge bestrahlt wurden, wurde bewertet, um die effektivsten Photoanregungsbedingungen zu finden. Die physikochemischen Eigenschaften und toxischen Wirkungen von C60 auf die Leukämiezellen wurden nach nicht kovalenter Bindung von Arzneistoffen bewertet. Schließlich wurden nanomolare Mengen von C60-Wirkstoff-Nanokomplexen in Molverhältnissen von 1:1 und 2:1 untersucht, um die Effizienz der Behandlung von Zellen zu verbessern und sie durch photodynamischen Ansatz zu ergänzen. Mit dem gängigen Chemotherapeutikum Doxorubicin wurde eine Behandlungsstrategie entwickelt und dessen intrazelluläre Akkumulation und Lokalisation, Zytotoxizität und Wirkmechanismus untersucht wurden. Es wurde gezeigt, dass die entwickelte Strategie auch auf ein alternatives Krebsmedikament übertragbar ist – das pflanzliche Alkaloid Berberin. Die erhaltenen Daten deuten darauf hin, dass eine Kombination von chemo- und photodynamischen Behandlungen mit C60-Nanokomplexen einen vielversprechenden synergetischen Ansatz für die Krebsbehandlung bieten könnte.

No description available.