Polyethylenimine-stabilized calcium phosphate nanoparticles with flagellin-cargo were coated with a silica shell to protect the protein inside (Figure 1). The morphology and the particle size distribution of unloaded and flagellin-loaded calcium phosphate nanoparticles were determined by dynamic light scattering (Table 1) and scanning electron microscopy.
Figure 1. Schematic representation of the structure of calcium phosphate nanoparticles, loaded with flagellin (SF) or an inactive flagellin mutant (ΔL).
Protein PDI Average size (DLS)/nm Zeta potential/mV none 0.147 194 22±5 SF 0.211 330 9±4 ΔL 0.260 385 10±4 PDI=Polydispersity index from dynamic light scattering; DLS=Dynamic light scattering.
Table 1. Colloid-chemical data of protein-functionalized calcium phosphate nanoparticles (standard deviations given after the average in parentheses).
The average hydrodynamic diameter was around 190 nm for unloaded nanoparticles and 330-380 nm for protein-loaded calcium phosphate nanoparticles, indicating some agglomeration in the latter case. Representative DLS curves of functionalized nanoparticles are shown in Figure 2. The unloaded calcium phosphate nanoparticles had a positive zeta potential of +22 mV. The protein-loaded nanoparticles had a zeta potential of +10 mV. In Figure 3, scanning electron micrographs of the protein-loaded calcium phosphate nanoparticles are shown. The particles had a spherical morphology with a diameter between 60 and 100 nm.
Figure 2. Dynamic light scattering data of calcium phosphate nanoparticles without and with flagellin (SF) or an inactive flagellin mutant (ΔL).
Figure 3. Scanning electron micrographs of calcium phosphate nanoparticles, loaded with either flagellin (SF) (A) or an inactive flagellin mutant (ΔL) (B). Note that the particles are agglomerated due to the drying process in the electron microscope. The primary particle size is between 60 and 100 nm.
To test the immunomodulating function of flagellin-functionalized nanoparticles, we tested whether they stimulated reagent DOTAP, SF induced the production of IL-1β by macrophages to a small extent. When SF was incorporated into nanoparticles, it induced IL-1β much more efficiently (Figure 4). However, it is remarkable that the unloaded calcium phosphate nanoparticles and the △L-loaded nanoparticles also led to a significant expression of IL-1β. This points to some general effect of stimulation, possibly by dissolved calcium ions after uptake or by the stabilizing agent poly (ethyleneimine).
Figure 4. Flagellin-loaded nanoparticles induced the production of proinflammatory cytokines in vitro. A: Human Caco-2 cells were stimulated with flagellin-functionalized nanoparticles or purified flagellin for 6 hours and supernatants were collected for IL-8 detection. B: Mice bone marrow-derived macrophages were pretreated with 50 ng mL-1 LPS for 3 hours and then stimulated with flagellin-functionalized nanoparticles or purified flagellin for 6 h, and supernatants were collected for IL-1β detection. The dashed line represents the control. SF = flagellin; ΔL = inactive flagellin mutant; SF-nano = nanoparticles, loaded with SF; △L-nano = nanoparticles, loaded with ΔL; SF+DOTAP = flagellin + transfection agent DOTAP; ΔL = inactive flagellin mutant + transfection agent DOTAP; nano = nanoparticles without protein.
The effects of flagellin-functionalized nanoparticles on innate immunity were further tested in vivo using IL-6 as the indicator. C57BL/6 mice were intraperitoneal immunized, and the serum and peritoneal lavage fluids (PLFs) were collected as described above. The contents of IL-6 in PLFs and serum were determined by ELISA. SF-loaded calcium phosphate nanoparticles more efficiently induced the production of IL-6 in serum in comparison to the dissolved SF protein alone. The only other system that led to an increased concentration of IL-6 in the serum was △L-loaded nanoparticles (Figure 5). Surprisingly, there were no significant differences between the levels of IL-6 in PLFs of mice treated with SF-loaded nanoparticles and that mice treated with △L-loaded nanoparticles. In the PLF, the nanoparticles alone also led to an increase in the IL-6 production, again pointing to a hitherto not understood effect of the nanoparticles alone.
Figure 5. Flagellin-functionalized nanoparticles induced proinflammatory cytokine productions in vivo. C57BL/6 mice were intraperitoneally immunized, and the serum and peritoneal lavage fluids (PLFs) were collected as described in materials and methods. The contents of IL-6 in serum (A) and PLFs (B) were determined by ELISA. SF = flagellin; ΔL = inactive flagellin mutant; SF-nano = nanoparticles, loaded with SF; ΔL-nano = nanoparticles, loaded with ΔL; nano = nanoparticles without protein.