We report the use of nano-meter sized zeolite particles as a novel approach to follow the endosomal acidification and proteolysis inside a viable cell. The method was verified by using human peripheral monocytes, a well known endocytosing cell population. Zeolite particles were subsequently used to investigate the endocytosing mechanisms of human peripheral dendritic cells (DCs). Probes detecting pH neutral and acidic endosomes were adsorbed to de-aluminated zeolite Y, and used to detect endocytosis in immature human peripheral blood DCs. Both the myeloid (mDCs) and the plasmacytoid (pDCs) dendritic cell subsets had an endocytosing capacity comparable to peripheral blood monocytes. However, the majority of both subsets of DCs retained their endosomes at a neutral pH during the first hours after endocytosis and only a small number of the mDCs showed any formation of acidic endosomes.Proteolytic degradation of endocytosed proteins was detected using self-quenched DQ-ovalbumin adsorbed to zeolite particles. Interestingly, a clear difference in proteolytic degradation of endocytosed ovalbumin was observed between the two subsets of DCs. The mDC showed an efficient degradation of ovalbumin, while the pDC population displayed no or only minor proteolytic degradation. In conclusion, zeolite particles provide a useful tool to study the endocytosing mechanisms, and an efficient carrier of bio-molecules into the endosomal pathways of viable cells.

Human peripheral dendritic cells (DCs) are antigen-presenting cells with the ability to internalize antigen and present antigen-derived peptides to T cells. Human DCs express several receptors on the surface for endocytosis and other recognition receptors that bind to microbes or microbial products, which are internalized and processed. Here, we report the use of nanometer-size zeolite particles as a tool to study receptor-mediated endocytosis by the two subsets of immature DCs, myeloid (mDC) and plasmacytoid (pDC) dendritic cells. A major difference in receptor-mediated endocytosis was observed between the two populations of peripheral DCs. The pDC population demonstrated an almost complete lack of receptor-mediated endocytosis of zeolite particles, whereas the mDC population demonstrated a clear receptor-mediated endocytosis. Fc receptors are expressed by both peripheral DC populations and lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are known ligands of the Toll-like receptor (TLR)-2 and TLR4, respectively, both TLRs expressed by human mDCs. An efficient receptormediated endocytosis of immunoglobulin G-, LTA-, and LPS-coated zeolite particles was observed by the mDC population and their endocytosing capacity depended strongly on the density of the ligand adsorbed onto the zeolite particles. In conclusion, an efficient receptor-mediated endocytosis was observed from the mDC population, whereas the pDCs demonstrated an almost complete lack of receptor-mediated endocytosis and nanometer-size dealuminated zeolite particles were a useful tool for studying receptor-mediated endocytosis in human peripheral DCs.

A unique property of dealuminated zeolite particles is the exceptional ability to bind both hydrophilic and hydrophobic biomolecules without any covalent linkages. By adsorbing phospholipids onto the particle surface, capture of particles by human peripheral myeloid dendritic cells (mDCs) was observed. Capture of zeolite particles was only seen when a low density of phosphatidylcholine was present on the particles, indicating a specific recognition of the structural features realised by phosphatidylcholine after adsorption on the particle. Adsorbing IgG on the particles revealed capture by mDCs that was dependent upon the density of the IgG molecules. To obtain a smaller particle exposing a high density of IgG molecules, immune complexes (ICs) were formed and both mDCs and pDCs (peripheral plasmacytoid DCs) captured immune complexes, although the mDCs showed a more efficient capture of ICs. As expected, mDCs captured and internalized ICs, whereas pDCs captured ICs but showed no internalization of ICs.