Orientador: Renato Atílio Jorge / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-17T02:06:37Z (GMT). No. of bitstreams: 1 Alves_JulianaMachadodaSilveira_M.pdf: 2543897 bytes, checksum: cfe682f5663f70da68e8234395efa7bc (MD5) Previous issue date: 2010 / Resumo: As porfirinas 5,10,15,20(3-hidroxifenil)porfirina (m-THPP), 5-hexil-10,20-bis(3hidrofenil)porfirina (hex-m-bisHPP) e 5-hexil-10,15,20-tris(3-hidroxifenil)porfirina (hex-m-triHPP), têm o mesmo rendimento quântico de oxigênio singleto e foram encapsuladas em nanopartículas (NPs) poli(láctico-co-glicólico) (PLGA) 50:50, preparadas através do método de emulsão e evaporação. As NPs obtidas têm diâmetros médios na faixa de submicrometros (<240 nm) e pequena dispersividade. O potencial zeta medido mostrou uma pequena variação (de -21.6 mV a -19.3 mV). A porcentagem de encapsulação das porfirinas obtidas foram: 93 ± 2; 97 ± 2 and 69 ± 1 para m-THPP, hex-m-bisHPP e hex-m-triHPP respectivamente. A eficácia fotodinâmica e a internalização das porfirinas foi investigada a 37oC utilizando células humanas cancerígenas de próstata (LNCaP). Após 2 horas de incubação com as NPs contendo a porfirina as porcentagens de internalização celular foram iguais para todas as porfirinas. As três porfirinas causam morte celular e dão a mesma viabilidade quando se varia o tempo de incubação (30-120 min), concentração da porfirina (2,5 a 7,5 mmol L) e dose de luz incidente (33 a 99 J cm). Análise das células por microscopia confocal mostrou que as porfirinas encapsuladas nas NPs foram localizadas no citoplasma, sempre na região perinuclear. Estes resultados mostram que porfirinas com estruturas semelhantes e anfifilicidades diferentes, com igual rendimento quântico de rendimento singleto e internalizadas nas células em concentrações iguais, tem a mesma eficácia fotodinâmica / Abstract: Porphyrins (5,10,15,20-tetra(3-hydroxyphenyl)porphyrin (m-THPP), 5-hexyl-10,20-bis(3-hydroxyphenyl) porphyrin (hex-m-bisHPP) and 5-hexyl-10,15,20-tris(3-hydroxyphenyl)porphyrin (hex-m-triHPP)) with different amphiphicities and equal singlet oxygen quantum yield in ethanol, were encapsulated into 50:50 poly(lactide-co-glycolide,) (PLGA) nanoparticles (NPs) prepared by the emulsion/evaporation technique. The NPs obtained had submicron average diameters (<240 nm) with low polydispersity. The zeta potential measurements showed slight variations in negativity (from -21.6 mV to -19.3 mV). Particle recovery (%) was determined with results of 93 ± 2; 97 ± 2 and 69 ± 1 for m-THPP, hex-m-bisHPP and hex-m-triHPP porphiryns, respectively. The photodynamic efficacy of the porphyrin-loaded nanoparticles and their cellular uptake at 37 oC was investigated with LNCaP prostate tumour cells. After 2 h incubation with porphyrin-loaded nanoparticles the percents of intracellular uptake were the same for all porphyrins. The three porphyrins cause cell death and gave the same cell viability with variations of incubation time (30¿120 min), drug concentration (2.5 to 7.5 mmol L) and incident light dose (33 to 99 J cm). Confocal laser scanning microscopy data showed that the porphyrin-loaded nanoparticles, were localized in the cells, always in the perinuclear region. These results show that porphyrins with similar structures and equal singlet oxygen quantum yields and internalized in equal concentrations in the cells, but different amphiphilicities, have equal photodynamic efficacy / Mestrado / Físico-Química / Mestre em Química

No description available.

The phenomenon of photobleaching of a photosensitizer during photodynamic therapy (PDT) is well known. For second generation photosensitizers it may be possible to exploit this effect to enhance the volume of damaged tissue and improve the efficacy of PDT. In addition, as a consequence of photobleaching, the fluorescence emitted by the photosensitizer will decrease during PDT. Mathematical models were developed which describe fluorescence emission, photobleaching and tissue necrosis resulting from the irradiation of tissue containing photosensitizer using an appropriate light source. Diffusion theory was used to model bleaching in a semi-infinite medium caused by broad-beam irradiation, and both pencil and broad-beam fluorescence excitation of the photosensitizer. In addition, models were developed for an isotropic point source imbedded in an infinite medium. Based on the relationship between the decreasing fluorescence signal and the increasing volume of tissue damage, these models allow the extent of necrosis achieved during treatment to be monitored. By analysing spatially resolved fluorescence measurements predictions about necrosis depth that are insensitive to treatment parameters such as photosensitizer concentration, tissue optical properties and bleach rate are possible. Tissue simulating optical phantoms that allow for relatively simple and accurate alteration to optical properties were developed. Photosensitizers which still undergo fluorescence and photobleaching in the solid medium were also added. Using these phantoms, treatment was simulated and spatially resolved fluorescence was measured as a function of time for a wide range of initial treatment parameters. Photobleaching of the photosensitizers was observed to occur through a decrease in fluorescence emission. Also, spatially resolved measurements provided information about the average photosensitizer depth, which was seen to increase with time, with little knowledge of initial treatment parameters. These experimental results were then compared with predictions from the mathematical theory, illustrating the validity of the models. The value and feasibility of this technique for photodynamic therapy dosimetry are discussed, along with planned improvements. / Thesis / Master of Science (MS)

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.

Photodynamic Therapy (PDT) has proven itself in previous studies to be a successful therapeutic treatment for surface tumors, but its effectiveness is limited to only shallow depths that allow for the penetration of light. This study demonstrates that we have improved upon the conventional method of PDT and have overcome the previous depth limitation by creating the light at the location of the tumor in situ. We conjugated a bioluminescent protein, Luciferase, to a semiconductor nanoparticle, TiO2, and with a cell specific antibody, anti-EGFR monoclonal antibody C225. The nanoconjugate, TiDoL-C225, was then activated by ATP and Luciferin in a reaction that creates reactive oxygen species (ROS) and induces apoptosis in the tumor cells. We created the optimal nanoconjugate synthesis protocol to make TiDoL and TiDoL-C225 for use in the PDT treatment. The TiDoL-C225 nanoconjugate is able to bind specifically to colon caner cells as the C225 antibody recognizes EGFR expressed at the surface of the cells, and further, when activated it will react only with the tumor cells. The optimal cell staining protocols were developed to visualize the treatment process and later analyze with the laser confocal microscope. The TiDoL nanoconjugate was found to only be operational and effective at killing tumor cells after being activated by Luciferin and ATP, which then enhances the control we have over the therapy. The TiDoL-C225 nanoconjugate increases the efficacy of binding to tumor cells and the speed of the reaction in the cells to begin apoptosis, even in lower concentrations when compared to the free TiDoL nanoconjugate. Finally, our PDT technique allowed us to monitor the tumor cells as they begin to undergo apoptosis in less than five minutes after the Luciferin was added to activate the reaction. The advantage of our method of PDT with the TiDoL-C225 nanoconjugate is that it can be used for early detection as well as developed into an effective treatment for cancers in all depths of tissue.

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.

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.