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

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

Gene Expression in Synovial Membrane Cells After Intraarticular Delivery of Plasmid-Linked Superparamagnetic Iron Oxide Particles—A Preliminary Study in Sheep

Alke Fink, Heinrich Hofmann, Benedikt Steitz

This study evaluated in vivo gene deliver y and subsequent gene expression within cells of the synovium in the presence of static and pulsating magnetic field application following intraar ticular injection of super paramagnetic iron oxide nanopar ticles linked to plasmids containing reporter genes encoding for fluorescent proteins. Plasmids encoding genes for either green fluorescent protein or red fluorescent protein were bound to super paramagnetic nanopar ticles coated with polyethyleneimine. Larger (200–250 nm) and smaller (50 nm) nanopar ticles were compared to evaluate the effects of size on transfection efficiency as well as any associated intraar ticular reaction. Comparisons between groups were evaluated at 24, 72, and 120 h time periods. Inflammator y response was mild to moderate for all injected par ticles, but was present in the majority of synovial membrane samples evaluated. Larger par ticles tended to be associated with more inflammation than smaller ones. Never theless, intraar ticular application of both experimental and control nanoparticles were well tolerated clinically. Gene expression as determined by obser vation of either green or red intracellular fluorescence was difficult to assess by both epifluorescent light, and confocal microscopy. An insufficient concentration of nanopar ticles in relation to joint volume likely resulted in a limited number of samples with positive evidence of iron staining and with suspected positive evidence of cells expressing fluorescent proteins. Our results indicate that intraar ticular administration of functionalized super paramagnetic iron oxide nanopar ticles resulted in a mild to moderate synovitis and there was in conclusive evidence of gene expression. Fur ther research is warranted to determine the best and most effective repor ter assay for assessment of the in vivo gene deliver y into the joints. In addition, the best suited concentration and size of nanopar ticles, which will optimize gene delivery and expression, while minimizing intraarticular inflammation, needs to be determined.

2006

Thrombosis efficacy induced by photodynamic therapy using different photosensitizers

Bernadette Pegaz-Toquet

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.

EPFL2007

Glycoside esters of 5-aminolevulinic acid for photodynamic therapy of cancer

Jérôme Barge, Frédéric Schmitt, Georges Wagnières

Aliphatic and ethylene glycol esters of 5-aminolevulinic acid (ALA) are very efficient precursors of the photosensitizer protoporphyrin IX (PpIX) for photodynamic therapy; however, they diffuse passively across the cell membrane and thus lack cell selectivity. We evaluated whether alpha-glucose, alpha-mannose, or beta-galactose esters of ALA would present improved properties as precursors of PpIX. Esterification was performed either at the position O-1 or O-6 of the sugars with or without an ethylene glycol linker, and these glycoside esters of ALA were evaluated in human cells. The results demonstrated that glycoside esters of ALA are efficient precursors of PpIX in human cancer and angiogenic endothelial cells, comparable to free ALA, but not in normal human fibroblasts. PpIX production was confirmed by fluorescence microscopy and photodynamic treatment of cells. The O-1 or O-6 positions of functionalization and the nature of the sugar moiety did not influence PpIX production. The presence of the ethylene glycol linker generally resulted in decreased PpIX production. The uptake of these glycoside esters of ALA by cells was not decreased in the presence of high concentrations of the related sugars. Inhibitors of alpha-glucosidases or alpha-mannosidases did not decrease PpIX production. These results suggest the involvement of active non-glycoside-specific membrane transporter(s) for uptake and of esterases rather than glycosidases in the release of ALA from the glycoside esters of ALA.