The effect of short silica fibers (0.3 µm 3.2 µm) on macrophages.

Silica fibers with a dimension of 0.3 µm ∙ 3.2 µm2 nm were prepared by a modified Stöber synthesis as model particles. The particles were characterized by scanning electron microscopy, elemental analysis, thermogravimetry and X-ray powder diffraction. Their uptake by macrophages (THP-1 cells and NR8383 cells) was studied by confocal laser scanning microscopy and scanning electron microscopy. The uptake by cells was very high, but the silica fibers were not harmful to NR8383 cells in concentrations up to 100 µg mL-1. Only above 100 µg mL-1, significant cell toxic effects were observed, probably induced by a high dose of particles that had sedimented on the cells and led to the adverse effects. The chemotactic response as assessed by the particle-induced migration assay (PICMA) was weak in comparison to a control of agglomerated silica particles. The as-prepared fibers were fully X-ray amorphous but crystallized to ß-cristobalite after heating to 1000 °C and converted to α-cristobalite upon cooling to ambient temperature. The fibers had sintered to larger aggregates but retained their elongated primary shape. The particle cytotoxicity towards THP-1 cells was not significantly enhanced by the crystallization.

Genetic immunization against hepatitis B virus with calcium phosphate nanoparticles in vitro and in vivo.

Calcium phosphate nanoparticles were loaded with plasmid DNA and toll-like receptor ligands (TLR), i.e. CpG or flagellin, to activate antigen-presenting cells (APCs) like dendritic cells (DCs). The functionalized nanoparticles were studied in vitro on HeLa, C2C12 and BHK-21 cell lines, focusing on the expression of two specific proteins. EGFP-DNA, encoding for enhanced green fluorescent protein (EGFP), was used as a model plasmid to optimize the transfection efficiency in vitro by fluorescence microscopy and flow cytometry. Calcium phosphate nanoparticles loaded with TLR ligands and plasmid DNA encoding for the hepatitis B virus surface antigen (pHBsAg) were evaluated by in vitro and in vivo immunization experiments to identify a possible candidate for a prophylactic hepatitis B virus (HBV) vaccine. The nanoparticles induced a strong expression of HBsAg in the three cell lines. In splenocytes, the expression of the co-stimulatory molecules CD80 and CD86 was enhanced. After intramuscular injection in mice, the nanoparticles induced the expression of HBsAg, the antigen-specific T cell response, and the antigen-specific antibody response (IgG1). STATEMENT OF SIGNIFICANCE: Hepatitis B is one of the most frequent viral infections worldwide. For preventive immunization, nanoparticles can be used which carry both an adjuvant (a stimulatory molecule) and DNA encoding for a viral antigen. After administration of such nanoparticles to cells, they are taken up by cells where the DNA is transcribed into the viral antigen (a protein). This viral antigen is inducing a virus-specific immune response. This was shown both by in vitro cell culture as well as by an extensive in vivo study in mice.

Calcium phosphate nanoparticles show an effective activation of the innate immune response in vitro and in vivo after functionalization with flagellin.

For subunit vaccines, adjuvants play a key role in shaping the magnitude, persistence and form of targeted antigen-specific immune response. Flagellin is a potent immune activator by bridging innate inflammatory responses and adaptive immunity and an adjuvant candidate for clinical application. Calcium phosphate nanoparticles are efficient carriers for different biomolecules like DNA, RNA, peptides and proteins. Flagellin-functionalized calcium phosphate nanoparticles were prepared and their immunostimulatory effect on the innate immune system, i.e. the cytokine production, was studied. They induced the production of the proinflammatory cytokines IL-8 (Caco-2 cells) and IL-1ß (bone marrow-derived macrophages; BMDM) in vitro and IL-6 in vivo after intraperitoneal injection in mice. The immunostimulation was more pronounced than with free flagellin.

Immunization with biodegradable nanoparticles efficiently induces cellular immunity and protects against influenza virus infection.

The ability of vaccines to induce T cell responses is crucial for preventing diseases caused by viruses or bacteria. Nanoparticles (NPs) are considered an efficient tool for inducing potent immune responses. In this study, we describe a novel vaccination approach with biodegradable calcium phosphate (CaP) NPs that serve as carrier of immunoactive TLR9 ligand (CpG) combined with a viral Ag from the influenza A virus hemagglutinin. Functionalized CaP NPs were efficiently taken up by dendritic cells in vivo and elicited a potent T cell-mediated immune response in immunized mice with high numbers of IFN-γ-producing CD4(+) and CD8(+) effector T cells. Most importantly, both i.p. and intranasal immunization with these NPs offered protection in a mouse model of influenza virus infection. This study demonstrates the great potential of CaP NPs as a novel vaccination tool that offers substantial flexibility for several infection models.

Synthetic pathways to make nanoparticles fluorescent.

In biosciences, it is often necessary to follow the pathway of nanoparticles within cells or tissues. The nanoparticles can be used as labeled sensors which may, e.g., address functionalities within a cell, carry other specific agents like drugs or be magnetic for tumor thermotherapy. In the context of nanotoxicology, the fate of a given nanoparticle is of interest. As many methods in cell biology are based on fluorescence detection, there is a strong demand to make nanoparticles fluorescent. Different ways to introduce fluorescence are reviewed and exemplified with typical kinds of nanoparticles, i.e. polymers, silica and calcium phosphate.