Photodynamic Therapy (PDT) has proven to be a minimally invasive alternative treatment option for various conditions including cancer. The treatment efficacy of deep-seated tumours with PDT is variable, compared to the treatment of tissue surfaces such as the skin and esophagus. This is partly due to inadequate monitoring of the three interrelated treatment parameters: treatment light, photosensitizer and tissue oxygenation. This thesis presents the development of a system for explicit dosimetry of PDT treatment light and tissue oxygenation using multi-sensor fiber optic probes for spatially resolved parameter measurements. The system uses embedded fluorescent sensors for treatment light quantification. Tissue oxygenation measurement is accomplished using frequency domain techniques with embedded phosphorescent metalloporphyrin compounds as sensors.

In photodynamic therapy (PDT), the combination of light, photosensitizer and molecular oxygen generates reactive oxygen species, including singlet oxygen (1O2), which is regarded as the primary cytotoxin and effector with most clinical photosensitizers. PDT has gained some acceptance for the treatment of cancer and other conditions. However, its clinical utility and effectiveness has been limited by variability in treatment response and failure to integrate adequate treatment planning and dosimetry. Direct PDT dosimetry through the detection of ultra-weak near-infrared 1O2 luminescence emission at 1270 nm (SOL) collapses the complexity of PDT into a single parameter, the 1O2 concentration. Prior to the present studies, it was shown that SOL was well correlated with PDT response in vitro and in vivo under controlled experimental conditions. However, SOL detection is technically challenging because of the very low radiative probability of 1O2 (~ 10-8 in biological environments), dynamic background signals and limited sensitivity of suitable photodetectors in this wavelength region. A technologically simpler and less costly PDT dosimetry approach is to use photosensitizer photobleaching to estimate the 1O2 dose. The first objective in this thesis was to characterize the dynamics of SOL measurements, in particular the influence of oxygen depletion, in order to improve the quantification of SOL and its use as an accurate PDT dose metric. Subsequently, direct comparison of SOL and photobleaching-based dosimetry during in vitro PDT treatment with meso-tetra(hydroxyphenyl)chlorin (mTHPC) showed that SOL dosimetry is robust but that photobleaching-based dosimetry can fail under hypoxic conditions. However, the latter can be salvaged through the identification of a previously unreported 613 nm emission from mTHPC that indicates hypoxia. These studies were carried forward into an in vivo dorsal skin-fold window chamber tumor model, which showed promising initial correlation between 1O2 dose and tumor response. This work also identified SOL detection limitations and opportunities for further development. Additionally, SOL measurements were used as a ‘gold standard’ to evaluate novel activatable PDT beacons and a novel “PDT biodosimeter” based on STAT3 cross-linking. Future work includes further tumor dose-response studies, characterization of novel photosensitizing agents, improvement on signal detection and processing, and studies in normal human skin.

In photodynamic therapy (PDT), the combination of light, photosensitizer and molecular oxygen generates reactive oxygen species, including singlet oxygen (1O2), which is regarded as the primary cytotoxin and effector with most clinical photosensitizers. PDT has gained some acceptance for the treatment of cancer and other conditions. However, its clinical utility and effectiveness has been limited by variability in treatment response and failure to integrate adequate treatment planning and dosimetry. Direct PDT dosimetry through the detection of ultra-weak near-infrared 1O2 luminescence emission at 1270 nm (SOL) collapses the complexity of PDT into a single parameter, the 1O2 concentration. Prior to the present studies, it was shown that SOL was well correlated with PDT response in vitro and in vivo under controlled experimental conditions. However, SOL detection is technically challenging because of the very low radiative probability of 1O2 (~ 10-8 in biological environments), dynamic background signals and limited sensitivity of suitable photodetectors in this wavelength region. A technologically simpler and less costly PDT dosimetry approach is to use photosensitizer photobleaching to estimate the 1O2 dose. The first objective in this thesis was to characterize the dynamics of SOL measurements, in particular the influence of oxygen depletion, in order to improve the quantification of SOL and its use as an accurate PDT dose metric. Subsequently, direct comparison of SOL and photobleaching-based dosimetry during in vitro PDT treatment with meso-tetra(hydroxyphenyl)chlorin (mTHPC) showed that SOL dosimetry is robust but that photobleaching-based dosimetry can fail under hypoxic conditions. However, the latter can be salvaged through the identification of a previously unreported 613 nm emission from mTHPC that indicates hypoxia. These studies were carried forward into an in vivo dorsal skin-fold window chamber tumor model, which showed promising initial correlation between 1O2 dose and tumor response. This work also identified SOL detection limitations and opportunities for further development. Additionally, SOL measurements were used as a ‘gold standard’ to evaluate novel activatable PDT beacons and a novel “PDT biodosimeter” based on STAT3 cross-linking. Future work includes further tumor dose-response studies, characterization of novel photosensitizing agents, improvement on signal detection and processing, and studies in normal human skin.

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.

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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.

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