Thrombosis efficacy induced by photodynamic therapy using different photosensitizers

Various human pathologies, such as cancers or, notably, exudative age-related macular degeneration (AMD), are characterized by an abnormal vascular proliferation (angiogenesis), which in the case of this type of AMD, can rapidly lead to blindness if no countermeasures are taken. In the past few years, it has become possible to stop - or at least to notably slow down - the evolution of AMD, by applying treatments based on photodynamic therapy (PDT), whereby, through the action of a molecule acting as photosensitizer (PS) which is targeted preferentially to the choroidal neovessels (CNV), the effects of this abnormal angiogenesis are stopped by provoking the closure of the neovessels. In particular, the PDT group at EPF-Lausanne has, in the past, already tested on the model of the chick embryo chorioallantoic membrane (known as the CAM model) several PSs of potential interest as substitute to the product currently considered as standard (Visudyne® , itself co-developed in this group) for the treatment of the CNV associated with AMD. The aim of this thesis was to study new molecules and/or formulations, potentially suitable as substitutes to the current photodynamic treatment of AMD, while diminishing its negative side-effects: general phototoxicity due to diffusion of the product out of the vasculature and further lack of selectivity leading to (undesirable) partial occlusion of the normal vasculature. In order to enable extrapolation and comparison of the results obtained, we have focused our attention on vascular effects only and administered the tested PSs under essentially similar conditions (formulation in DMSO; same delivery system through biodegradable nanoparticles built with the same polymer). In addition, the same animal model (the CAM model) was used throughout the entire study, the formulations being administered by intravenous injection. This model, importantly, allows for an easy and real-time semi-quantitative visualisation and recording of the vascular effects of a PS induced during a PDT session. In the first part of this work, we demonstrate that the tetra-phenylporphyrin molecule, when formulated in nanoparticles based on poly(D, L - lactic acid, of well defined size (ca. 200 nm or less) is a very interesting PS-candidate potentially suitable as a replacement of the currently used one. We show that inclusion of this PS in nanoparticles notably reduces the PDT's general phototoxicity. We believe therefore that this PS would deserve more thorough pre-clinical studies, on an animal model closer to the human case (for example, Ryan's model). The second part of this thesis aimed at elaborating a strategy allowing, in due time, for an improved selectivity of occlusion of the neovasculature, said strategy aiming at an active targeting of neovessels. Recently, a number of possible biochemical targets have been identified, including the so-called ανβ3 receptors, which are highly expressed at the level of the neovessels' endothelial cells. Our approach consisted in coupling a fluorescent molecule (fluorescein, easily visualized by epi-microscopy) to a protein (a kistrin) which contains the RGD peptide sequence, known for its affinity for the ανβ3 receptors. We have thus been able to demonstrate that the kistrin was effectively linked to these receptors and, therefore, that active targeting of the neovessels had indeed taken place. A complete proof of this concept by realizing the coupling of this kistrin with a PS molecule will be the next step in this research.

Lipid membranes for the fabrication of functional micro- and nano-structures

Gopakumar Gopalakrishnan

The central goal of this thesis work is to fabricate novel, functional fluorescent nanostructures in confined systems offered by phospholipid membranes, which are known to have highly ordered, thermotropic and lyotropic structures. In separate approaches, we have used three different lipid systems: multilamellar planar lipid membranes, unilamellar vesicular membranes as well as lipid monolayers for the development of functional fluorescent nano-, micro- and meso-scopic structures. Techniques like fluorescence microscopy, single particle imaging, electron microscopy, electron diffraction were used to achieve fundamental understanding of the resulting structures. Multilayer stacks of phospholipid membranes have been used as an effective template for the growth of high aspect ratio fluorescent nanowires. The room temperature synthesis was achieved in the confined nanometer-sized interlamellar water space of lipid multilayers where supersaturating CdCl2 concentrations were induced by acidification leading to controlled unidirectional growth of nanowires. The possibility to render the nanowires fluorescent by doping with CdS quantum dots (QDs) and the light waveguiding along hundreds of micrometers together with the possibility of lateral manipulation make these nanowires attractive candidates for future optoelectronic applications. Novel organic-inorganic functional nanocontainers have been designed and tested by making use of vesicle forming lipid bilayers in combination with semiconductor QDs. Hydrophobic QDs can be integrated into bilayers of lipid vesicles and such lipid/QD hybrid vesicles are capable to fuse with live cells, thereby stain the cell's plasma membrane selectively with fluorescent QDs and transfer the vesicle's cargo into the cell. Modification of the membrane of such hybrid vesicles on the other hand, made them capable to enter the cytoplasm of live cells. Additionally, these hybrid vesicles were found extremely useful for long-term model membrane imaging studies. The results described in this thesis imply that cell and lipid membranes can integrate any kind of hydrophobic nanoparticle whose size matches the membrane thickness, opening novel possibilities to manipulate them as individuals or in ensemble with wide-ranging applications for nanobiotechnology. In a further step, the ability of phospholipid molecules to exhibit lamellar to non-lamellar transition using external stimuli enabled a directed self-assembly of QDs into mesoscale fluorescent structures. An easy and versatile method for the surface modification of TOPO coated CdSe QDs using 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) have been achieved. DPPA predominantly form non-lamellar phases when dispersed in water, for example, upon addition of Ca2+ or at a pH below 6 are known to form hexagonal II phases. This particular property of DPPA has been exploited to form mesoscale self-assemblies of QD based structures both in solution and in confined systems. Potential applications include the detection or removal of Ca2+ ions in attoliter volumes, the construction of functional devices where QDs are reversibly organized in different forms as well as use of fluorescently labeled DPPA molecules for cellular studies using fluorescence microscopy.

EPFL2006

Development of Minimally-Invasive Optical Methods to Individualize the Doses Used for Therapeutic Applications of Light

Filippo Piffaretti

Despite the experience gained over several decades in various types of light-based medical treatments, the optimization of the corresponding therapeutic protocols and accurate forecasting of their outcome have not yet been achieved in many cases. The difficulty often arises from the heterogeneity of living tissues, their variable optical properties, and from the heterogeneous distribution of the photoactive or photosensitive substances – whether naturally present in the tissue or artificially adde. Our work focuses on the individualization and control of irradiation parameters, in order for the physician to be able to elicit a predictable clinical response in the irradiated tissues. In this thesis, we present three separate studies in which we tried to evaluate the possibility of individualizing and optimizing the corresponding clinical outcomes by measuring or monitoring certain, particular parameters. In a first clinical study, performed at the medical practice of Dr Vezzola, MD, in Saló, Italy, the human eye's retinal reflectance was measured and mapped, in the framework of subthreshold thermal laser therapy, using an excitation wavelength identical to that of the treatment laser, i.e. at 810 nm. The specific goal of this study was to correlate the occurrence of retinal burns with the measured infrared retinal reflectance. This study was performed using a modified fundus camera to record infrared reflectance images of the retina, and by recording the slit-lamp based laser therapy parameters (irradiation parameters and spot location) in such a way so as to overlay the map of the laser treatment spots on the corresponding reflectance fundus image. The clinical study demonstrated the expected existence of spatial variations in light reflectance at 810 nm (probably due to changes of the tissue absorption), which we then tried to relate to the occurrences of retinal burns observed during the laser treatment. The analysis of the results obtained with the applied conditions, did not however show a clear correlation between the local retina reflectance, the laser beam parameters, and the occurrence of retinal burns. Therefore we postulate that either the absorbing structures of the retina cannot be seen with the imaging device we used, possibly due to its limited resolution, or other important elements play a role in the laser-tissue interactions during this type of lasers-light interaction with the local retinal tissue. The second study was pre-clinical, performed at EPFL, and aimed at monitoring in real-time the tissular oxygen concentration during photodynamic therapy (PDT). It was performed in vivo on the chicken embryo's chorio-allantoic membrane (CAM) model, which was submitted to aminolevulinic acid (ALA)-based PDT. The molecular oxygen, which is thought to be an essential actor in the cascade of reactions leading to the tissular PDT effect, is actually the main molecule responsible for the photosensitizer's (PS) triplet state quenching. Therefore, the delayed fluorescence lifetime of the photosensitizer, protoporphyrin IX (PpIX), was measured with a specially designed and assembled, optical fiber-based, time-resolved spectrofluorometer, and used as a proxy for tissular pO2. Simultaneously, vascular damages caused by PDT were characterized and quantified, to check for correlation between the two parameters. Using the PS's delayed fluorescence lifetime to evaluate tissular pO2 proved to be a quite reasonable strategy, due to the fact that it is possible to measure the pO2 at the location of the PS molecule. The study's results demonstrate a robust, linear correlation between tissular pO2 reduction and vascular damage extent. They also suggest that the amount of oxygen consumed during PDT could be a useful, measurable parameter for assessing and/or controlling the PDT's therapeutic effect. The third, clinical, study aimed at measuring the fluorescence photobleaching of the PpIX photosensitizer due to PDT. This study was performed at the "Hôpitaux Universitaires de Genève – HUG", in Geneva, Switzerland, in collaboration with Dr Denis Salomon, MD, and Dr Behrooz Kasraee, MD, in the framework of a series of standard clinical PDT treatments of aktinic keratoses (AK), which is a pre-cancerous skin lesion. The lesions' fluorescence intensity was quantified with a specially adapted quantitative imaging device, using a homogeneous and constant intensity fluorescence excitation light. The specific goal was to check for a relation between the extent of photobleaching of the PS (in this case PpIX, which was induced by the administration of Metvix®) and the clinical outcome (disappearance of AK) evaluated several months after the treatment. The study's results show that the amount of photobleached PS is strongly and linearly correlated to the fluorescence measured before the treatment. Likewise, the preliminary assessment of the clinical outcomes confirms the existence of a correlation of these outcomes with the PS's fluorescence bleaching, thus making it possible, in principle, to select and optimize the PDT's irradiation parameters before starting the treatment. The results of this study demonstrate the relevance of measuring the PS's photo-bleaching for optimizing PDT and forecasting of its outcome.

EPFL2010

Chitosan-based nanogels for selective delivery of photosensitizers to macrophages and improved retention in and therapy of articular joints

Jérôme Barge, Lucienne Lagopoulos, Frédéric Schmitt, Georges Wagnières, Christine Wandrey

Macrophages play key roles in inflammatory disorders. Therefore, they are targets of treatments aiming at their local destruction in inflammation sites. However, injection of low molecular mass therapeutics, including photosensitizers, in inflamed joints results in their rapid efflux out of the joints, and poor therapeutic index. To improve selective uptake and increase retention of therapeutics in inflamed tissues, hydrophilic nanogels based on chitosan, of which surface was decorated with hyaluronate and which were loaded with one of three different anionic photosensitizers were developed. Optimal uptake of these functionalized nanogels by murine RAW 264.7 or human THP-1 macrophages as models was achieved after