In vitro uptake of gelatin nanoparticles by murine dendritic cells and their intracellular localisation

The long term goal Of this Study is to develop an efficient nanoscopic vaccine delivery system, based on the biodegradable and natural polymer gelatin, to deliver therapeutic protein antigens along with adjuvants into dendritic cells (DCs). In this study, gelatin nanoparticles were tested for qualitative and quantitative uptake in murine DCs in vitro. A second aim of this study was to prove that the carrier system is able to deliver tetramethylrhodamine Conjugated dextran (TMR-dextran), as a model drug into the DCs. The TMR-dextran was incorporated during the preparation of the gelatin nanoparticles. DCs were generated from murine bone marrow cells by an established ex vivo technique. Flow cytometry showed that 88% of the cells positive for the specific murine DC marker CD I I c took up TMR-dextran loaded gelatin nanoparticles, whereas only 4% of the soluble form of TMR-dextran was taken Lip. Double color confocal laser scanning microscopy (CLSM) showed that gelatin nanoparticles were phagocytosed by DCs and the triple color CLSM showed that the TMR-dextran was localized mainly in lysosomes as expected, but partly also outside the lysosomes, presumably in the cytoplasm. An in vitro release study of TMR-dextran from gelatin nanoparticles demonstrated that there was hardly any release in phosphate buffered saline (PBS), but by trypsin-assisted degradation of gelatin nanoparticles resulted in the release of about 80% of the TMR-dextran from the particles. These results Suggest that gelatin nanoparticles hold promise as a new biocompatible tool for vaccine delivery to DCs, with applications in cancer immunotherapy. (c) 2006 Elsevier B.V. All rights reserved.