A systematic electron microscopic study on the uptake of barium sulphate nano-, submicro-, microparticles by bone marrow-derived phagocytosing cells.

Nanoparticles can act as transporters for synthetic molecules and biomolecules into cells, also in immunology. Antigen-presenting cells like dendritic cells are important targets for immunotherapy in nanomedicine. Therefore, we have used primary murine bone marrow-derived phagocytosing cells (bmPCs), i.e. dendritic cells and macrophages, to study their interaction with spherical barium sulphate particles of different size (40 nm, 420 nm, and 1 µm) and to follow their uptake pathway. Barium sulphate is chemically and biologically inert (no dissolution, no catalytic effects), i.e. we can separate the particle uptake effect from potential biological reactions. The colloidal stabilization of the nanoparticles was achieved by a layer of carboxymethylcellulose (CMC) which is biologically inert and gives the particles a negative zeta potential (i.e. charge). The particles were made fluorescent by conjugating 6-aminofluoresceine to CMC. Their uptake was visualized by flow cytometry, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and correlative light and electron microscopy (CLEM). Barium sulphate particles of all sizes were readily taken up by dendritic cells and even more by macrophages, with the uptake increasing with time and particle concentration. They were mainly localized inside phagosomes, heterophagosomes, and in the case of nanoparticles also in the nearby cytosol. No particles were found in the nucleus. In nanomedicine, inorganic nanoparticles from the nanometer to the micrometer size are therefore well suited as transporters of biomolecules, including antigens, into dendritic cells and macrophages. The presented model system may also serve to describe the aseptic loosening of endoprostheses caused by abrasive wear of inert particles and the subsequent cell reaction, a question which relates to the field of nanotoxicology. STATEMENT OF SIGNIFICANCE: The interaction of particles and cells is at the heart of nanomedicine and nanotoxicology, including abrasive wear from endoprostheses. It also comprises the immunological reaction to different kinds of nanomaterials, triggered by an immune response, e.g. by antigen-presenting cells. However, it is often difficult to separate the particle effect from a chemical or biochemical reaction to particles or their cargo. We show how chemically inert barium sulphate particles with three different sizes (nano, sub-micro, and micro) interact with relevant immune cells (primary dendritic cells and macrophages). Particles of all three sizes are readily taken up into both cell types by phagocytosis, but the uptake by macrophages is significantly more prominent than that by dendritic cells. The cells take up particles until they are virtually stuffed, but without direct adverse effect. The uptake increases with time and particle concentration. Thus, we have an ideal model system to follow particles into and inside cells without the side effect of a chemical particle effect, e.g. by degradation or ion release.

Quantitative determination of the composition of multi-shell calcium phosphate-oligonucleotide nanoparticles and their application for the activation of dendritic cells.

Biodegradable calcium phosphate nanoparticles as carriers for the immunoactive toll-like receptor ligands CpG and polyinosinic-polycytidylic acid for the activation of dendritic cells (DC) combined with the viral antigen hemagglutinin (HA) were prepared. A purification method based on ultracentrifugation and ultrasonication was developed to separate the nanoparticles from dissolved biomolecules. The number of biomolecules, i.e., oligonucleotides and peptide, incorporated into the nanoparticles was quantitatively determined by UV-spectroscopy, using fluorescent derivatives of the biomolecules. The immunostimulatory effects of purified calcium phosphate nanoparticles on DC were studied, i.e., cytokine production and activation of the cells in terms of the upregulation of surface molecules. Purified calcium phosphate nanoparticles, i.e., without dissolved biomolecules, are capable of inducing adaptive immunity by activation of DC. Immunostimulatory effects of purified calcium phosphate nanoparticles on DC were demonstrated by increased expression of co-stimulatory molecules and MHC II and by cytokine secretion. In addition, DC treated with purified functionalized calcium phosphate nanoparticles induced an antigen-specific T-cell response in vitro.