Quantitative analysis of receptor-mediated uptake and pro-apoptotic activity of mistletoe lectin-1 by high content imaging.

Ribosome inactivating proteins (RIPs) are highly potent cytotoxins that have potential as anticancer therapeutics. Mistletoe lectin 1 (ML1) is a heterodimeric cytotoxic protein isolated from European Mistletoe and belongs to RIP class II. The aim of this project was to systematically study ML1 cell binding, endocytosis pathway(s), subcellular processing and apoptosis activation. For this purpose, state of the art cell imaging equipment and automated image analysis algorithms were used. ML1 displayed very fast binding to sugar residues on the membrane and energy-dependent uptake in CT26 cells. The co-staining with specific antibodies and uptake blocking experiments revealed involvement of both clathrin-dependent and -independent pathways in ML1 endocytosis. Co-localization studies demonstrated the toxin transport from early endocytic vesicles to Golgi network; a retrograde road to the endoplasmic reticulum. The pro-apoptotic and antiproliferative activity of ML1 were shown in time lapse movies and subsequently quantified. ML1 cytotoxicity was less affected in multidrug resistant tumor cell line 4T1 in contrast to commonly used chemotherapeutic drug (ML1 resistance index 6.9 vs 13.4 for doxorubicin; IC50: ML1 1.4 ng/ml vs doxorubicin 24000 ng/ml). This opens new opportunities for the use of ML1 as an alternative treatment in multidrug resistant cancers.

Dexamethasone-loaded poly(ε-caprolactone)/silica nanoparticles composites prepared by supercritical CO2 foaming/mixing and deposition.

A supercritical carbon dioxide (scCO2)-assisted foaming/mixing method (SFM) was implemented for preparing dexamethasone (DXMT)-loaded poly(ε-caprolactone)/silica nanoparticles (PCL/SNPs) composite materials suitable for bone regeneration. The composites were prepared from PCL and mesoporous SNPs (MCM-41/SBA-15) by means of scCO2-assisted SFM at several operational pressures, processing times and depressurization conditions. DXMT was loaded into SNPs (applying a scCO2 solvent impregnation/deposition method - SSID) and into PCL/SNPs composites (using the SFM method). The effects of the employed operational and compositional variables on the physicochemical and morphological features as well as in the in vitro release profiles of DXMT were analyzed in detail. This work demonstrates that the above-referred scCO2-based methods can be very useful for the preparation of DXMT-loaded PCL/SNPs composites with tunable physicochemical, thermomechanical, morphological and drug release properties and suitable for hard-tissue regeneration applications.