Comparing the Colloidal Stabilities of Commercial and Biogenic Iron Oxide Nanoparticles That Have Potential In Vitro/In Vivo Applications

Abstract

For the potential in vitro/in vivo applications of magnetic iron oxide nanoparticles, their stability in different physiological fluids has to be ensured. This important prerequisite includes the preservation of the particles' stability during the envisaged application and, consequently, their invariance with respect to the transfer from storage conditions to cell culture media or even bodily fluids. Here, we investigate the colloidal stabilities of commercial nanoparticles with different coatings as a model system for biogenic iron oxide nanoparticles (magnetosomes) isolated from magnetotactic bacteria. We demonstrate that the stability can be evaluated and quantified by determining the intensity-weighted average of the particle sizes (Z-value) obtained from dynamic light scattering experiments as a simple quality criterion, which can also be used as an indicator for protein corona formation.

Keywords: colloidal stability; magnetic nanoparticles; magnetosomes; protein corona.

Figure 1

Transmission electron micrographs and DLS results for different types of iron oxide nanoparticles. (a) TEM image of biogenic magnetosomes isolated from M. gryphiswaldense (b-NPs). (b) TEM image of commercial, citric acid encapsulated iron oxide nanoparticles (c-NPs). (c) TEM image of commercial phospholipid encapsulated iron oxide nanoparticles (p-NPs). Scale bar for TEM micrographs (a–c): 100 nm; scale bar insets: 50 nm. (d) Average diameter d and zeta potential ζ for the different iron oxide nanoparticle suspensions in water.

Figure 2

DLS results for iron oxide nanoparticle suspensions. (a) Stability of b-NP, c-NP and p-NP suspensions in RPMI and DMEM. (b–d) Z-mean values for c-NPs and p-NPs in aqueous solutions of inositol ((b), structure as inset), NaCl ((c), for full graph see Figure S4) and MgSO₄ (d) with various concentrations. The dashed lines show the limits for the metastable (short-dashed line) and unstable regions (long-dashed line).

Figure 3

TEM micrographs of different HSA-incubated magnetic NPs. Suspensions of b-NPs (left), c-NPs (middle) or p-NPs (right) (each 826 µg Fe mL⁻¹) were incubated with HSA at a final concentration of 10 mg mL⁻¹. After removal of excess HSA by performing several washing steps, the particles were analyzed by TEM. For b-NPs, a clear electron-light organic shell had already become visible in the unstained state (top left, indicated by arrows), suggesting the presence of a protein corona. In negatively stained preparations (bottom left), an even more pronounced well-defined shell could be visualized. In negatively stained samples of c-NPs and p-NPs (each incubated with HSA), an electron-light organic shell became visible as well (blue arrows); however, due to the rather irregular shape of the NPs and their strong tendency to form clusters, the layer thickness cannot easily be assessed in this case. (Scale bar: 100 nm, scale bar inset “c-NP + HSA”: 50 nm).

Figure 4

Overview of the Z-means and the different size classes in the investigated iron oxide NP suspensions (in HEPES or water). Overall, the NP stability in HEPES is higher than in water, and the number of larger species increases with each washing step. The data on p-NPs (a) and c-NPs (b) showed a similar trend; however, the latter are more stable compared with p-NP suspensions.

Figure 5

SDS-PAGE results for iron oxide nanoparticle suspensions in HEPES after addition of HSA (10 mg mL^–1). “M” denotes the lane of the protein molecular weight marker (“prestained plus”); the washing steps are indicated by their respective numbers. “S” indicates the supernatants and “P” the pellet of each sample. (a) The protein corona surrounding p-NPs was mostly rinsed off after the first washing step. Consequently, more HSA was found in the supernatant (1-S). The presence of a small band in the pellets even after several washing steps hints to the existence of a hard HSA protein corona (1-P, 2-P and 4-P). (b) Similar results were obtained for c-NPs.