Investigation of Aggregation and Disaggregation of Self-Assembling Nano-Sized Clusters Consisting of Individual Iron Oxide Nanoparticles upon Interaction with HEWL Protein Molecules

Abstract

In this paper, iron oxide nanoparticles coated with trisodium citrate were obtained. Nanoparticles self-assembling stable clusters were ~10 and 50-80 nm in size, consisting of NPs 3 nm in size. The stability was controlled by using multi-angle dynamic light scattering and the zeta potential, which was -32 ± 2 mV. Clusters from TSC-IONPs can be destroyed when interacting with a hen egg-white lysozyme. After the destruction of the nanoparticles and proteins, aggregates are formed quickly, within 5-10 min. Their sizes depend on the concentration of the lysozyme and nanoparticles and can reach micron sizes. It is shown that individual protein molecules can be isolated from the formed aggregates under shaking. Such aggregation was observed by several methods: multi-angle dynamic light scattering, optical absorption, fluorescence spectroscopy, TEM, and optical microscopy. It is important to note that the concentrations of NPs at which the protein aggregation took place were also toxic to cells. There was a sharp decrease in the survival of mouse fibroblasts (Fe concentration ~75-100 μM), while the ratio of apoptotic to all dead cells increased. Additionally, at low concentrations of NPs, an increase in cell size was observed.

Keywords: aggregation; fibroblast cell viability; hen egg-white lysozyme; toxicity; trisodium citrate–coated iron oxide nanoparticles.

Figure 5

(a) Fluorescence for HEWL (0.4 and 5 mg/mL), HEWL with TSC (1 M), and HEWL with TSC-IONPs (10¹³ NPs/mL). The points show the positions of the maxima. The intensities and coordinates of the maxima are written at the top of the graph. (b) Normalized emission of HEWL samples of various concentrations upon the addition of TSC-IONPs. (c–f) Emission of 0.01, 0.4, 5, and 100 mg/mL HEWL samples with different concentrations upon the addition of TSC-IONPs. Results are presented as mean and SD for three independent experiments.

Figure 1

Distribution (weighted by intensity) of hydrodynamic diameters of molecules, clusters, and aggregates for TSC-IONPs 10¹³ mL⁻¹, TSC 1 M, and HEWL (5 mg/mL) solution without NPs and TSC. Polydispersity index for TSC-IONPs 0.34 ± 0.15 (mean ± SD), for TSC 0.68 ± 0.16, and for HEWL 1.03 ± 0.17.

Figure 2

TEM-images: (a) large clusters of TSC-IONP nanoparticles, (b) small clusters of TSC-IONPs, (c) TSC-IONPs (10¹³ mL⁻¹) with HEWL (5 mg/mL), (d) the distribution of the lengths of the segments connecting the boundary pixels of the NPs in the TEM images (b,c). The lines show the Gaussian approximation of the distributions of segments for NPs in the clusters (green line, (b)) and NPs in the aggregates with HEWL (red line, (c)).

Figure 3

(a) EDS analysis of distribution of carbon (left top), oxygen (left bottom), iron (right top) in the sample (right bottom) TSC-IONPs (10¹³ mL⁻¹) with HEWL (5 mg/mL); (b) energy dispersive spectroscopy results for TSC-IONPs with HEWL.

Figure 4

(a) Photo of aggregates of HEWL (5 mg/mL) and TSC-IONPs (10¹³ mL⁻¹) made with a microscope. (b) Absorption of HEWL solution (0.4 mg/mL) with the addition of various concentrations of TSC-IONPs (up to 10¹³ mL⁻¹—red line) and TSC 1 M (purple line). Measurements were made relative to water for protein, or relative to the respective concentrations of TSC-IONPs and TSC. The top right inset shows absorption measurements of TSC-IONPs and TSC 1 M solutions concerning water.

Figure 6

Distribution (weighted by intensity) of hydrodynamic diameters of molecules and aggregates for different concentrations of NPs (from 10¹¹ to 5 × 10¹³ mL⁻¹) and different concentrations of HEWL: 0.01 (a), 0.4 (b), 5 (c) mg/mL. (d) Dependence of the average size of hydrodynamic diameter on the concentration of NPs (* p < 0.05). Polydispersity index for all measurements in the Supplementary Materials (Figure S7). Results are presented as mean and SD for five independent experiments.

Figure 7

(a) Time dependence of the average size of hydrodynamic diameter of HEWL (5 mg/mL) for TSC-IONPs (10¹² and 10¹³ mL⁻¹). Results are presented as mean and SD for three independent experiments. (b) DLS intensity data for solution HEWL (10 mg/mL) with TSC-IONPs (10¹² mL⁻¹): HEWL control, immediately after the addition of NPs, after 10 min, after 20 min; in addition, the sample was shaken for 10 s on a Biosan V1-plus vortex. Mean and SD were calculated from five measurements.

Figure 8

Rhodamine intensity in a fibroblast cell, the area occupied by a fibroblast cell relative to the entire frame area, and fibroblast cell viability after 24 h of exposure to TSC-IONPs of various concentrations. Results are presented as mean and SD for three independent experiments.

Figure 9

Representative images showing fibroblast cells at various concentrations of addition of iron oxide nanoparticles 24 h after exposure: (a) control cells, (b) cells with TSC-IONPs 5 × 10¹² mL⁻¹, (c) cells with TSC-IONPs 7.5 × 10¹² mL⁻¹, (d) cells with TSC-IONPs 10¹³ mL⁻¹. Staining with three types of fluorescent dyes: Hoechst 33342 (blue, stains the live cell nucleus), cell nuclei PI (red, stains the nucleus of dead cells), rhodamine-123 (green, mitochondrial membrane potential indicator).

Figure 10

The ratio of apoptotic cells (colored green with AF488-Annexin V+PI+) to the total number of dead cells (colored purple PI+) (a), a sample photo of the concentration of TSC-IONPs 7.5 × 10¹² mL⁻¹ (b).

Figure 11

Dependence of the relative value of the magnetic interaction $U/kT$ on the radius $r$ of two contacting IONPs without shells and at different shell thicknesses $d$: 1 nm for the citrate shell and 4 nm for the protein shell.