The intracellular uptake of CD95 modified paclitaxel-loaded poly(lactic-co-glycolic acid) microparticles.

The CD95/CD95L receptor-ligand system is mainly recognised in the induction of apoptosis. However, it has also been shown that CD95L is over-expressed in many cancer types where it modulates immune-evasion and together with its receptor CD95 promotes tumour growth. Here, we show that CD95 surface modification of relatively large microparticles >0.5 µm in diameter, including those made from biodegradable polylactic-co-glycolic acid (PLGA), enhances intracellular uptake by a range of CD95L expressing cells in a process akin to phagocytosis. Using this approach we describe the intracellular uptake of microparticles and agent delivery in neurons, medulloblastoma, breast and ovarian cancer cells in vitro. CD95 modified paclitaxel-loaded PLGA microparticles are shown to be significantly more effective compared to conventional paclitaxel therapy (Taxol) at the same dose in subcutaneous medulloblastoma (∗∗∗P < 0.0001) and orthotopic ovarian cancer xenograft models where a >65-fold reduction in tumour bioluminescence was measured after treatment (∗P = 0.012). This drug delivery platform represents a new way of manipulating the normally advantageous tumour CD95L over-expression towards a therapeutic strategy. CD95 functionalised drug carriers could contribute to the improved function of cytotoxics in cancer, potentially increasing drug targeting and efficacy whilst reducing toxicity.

The phagocytic capacity of neurones.

Phagocytosis is defined as the ingestion of particulates over 0.5 microm in diameter and is associated with cells of the immune system such as macrophages or monocytes. Neurones are not generally recognized to be phagocytic. Using light, confocal, time-lapse and electron microscopy, we carried out a wide range of in-vitro and in-vivo experiments to examine the phagocytic capacity of different neuronal cell types. We demonstrated phagocytosis of material by neurones, including cell debris and synthetic particles up to 2.8 microm in diameter. We showed phagocytosis in different neuronal types, and demonstrated that debris can be transported from neurite extremities to cell bodies and persist within neurones. Flow cytometry analysis demonstrated the lack of certain complement receptors on neurones but the presence of others, including integrin receptors known to mediate macrophage phagocytosis, indicating that a restricted set of phagocytosis receptors may mediate this process. Neuronal phagocytosis occurs in vitro and in vivo, and we propose that this is a more widespread and significant process than previously recognized. Neuronal phagocytosis may explain certain inclusions in neurones during disease, cell-to-cell spread of disease, neuronal death during disease progression and provide a potential mechanism for therapeutic intervention through the delivery of particulate drug carriers.

Culture of human keratinocytes on polypyrrole-based conducting polymers.

Variously loaded polypyrrole films, including those containing proteins and polysaccharides, were prepared on gold-coated polycarbonate coverslips. The characteristics of human keratinocytes were studied on these films by microscopy, biochemical assays, and immunocytochemistry. We found keratinocyte viability to be load dependent. For chloride, polyvinyl sulphate, dermatan sulphate, and collagen-loaded polypyrrole films, keratinocyte viability as assessed by the AlamarBlue assay was respectively 47.22, 60.43, 87.71, and 22.65% of tissue culture polystyrene controls after 5 days. This was found to require a previously unreported polymer washing step prior to cell seeding due to the observed toxicity of untreated films. In the case of bare polycarbonate and gold substrates, viability was respectively 75.44 and 61.04% of tissue culture polystyrene controls after 5 days. Keratinocytes stained positive for PCNA (proliferation), K10 (suprabasal differentiation), and K16 (hyperproliferation) markers although cell morphology was poor for organotypical cultures on dermatan- loaded polypyrrole compared with de-epidermalized dermis. From our studies, we concluded that optimized polypyrrole films adequately support keratinocyte growth in submerged cultures with some improvements needed for organotypical cultures. Polypyrrole composites are attractive candidates for tissue-engineering applications since they may incorporate biomolecules and are electrically addressable with the potential to both direct and report on cell activity.