Influence of PEG Chain Length of Functionalized Magnetic Nanoparticles on the Cytocompatibility and Immune Competence of Primary Murine Macrophages and Dendritic Cells

Abstract

A major drawback of nanoparticles (NPs) for biomedical applications is their preferential phagocytosis in immune cells, which can be avoided by surface modifications like PEGylation. Nevertheless, examinations of different polyethylene glycol (PEG) chain lengths on the competence of immune cells as well as possible immunotoxic effects are still sparse. Therefore, primary murine macrophages and dendritic cells were generated and incubated with magnetic nanoporous silica nanoparticles (MNPSNPs) modified with different mPEG chains (2 kDa, 5 kDa, and 10 kDa). Cytotoxicity, cytokine release, and the formation of reactive oxygen species (ROS) were determined. Immune competence of both cell types was examined and uptake of MNPSNPs into macrophages was visualized. Concentrations up to 150 µg/mL MNPSNPs showed no effects on the metabolic activity or immune competence of both cell types. However, ROS significantly increased in macrophages incubated with larger PEG chains, while the concentration of cytokines (TNF-α and IL-6) did not indicate a proinflammatory process. Investigations on the uptake of MNPSNPs revealed no differences in the onset of internalization and the intensity of intracellular fluorescence. The study gives no indication for an immunotoxic effect of PEGylated MNPSNPs. Nevertheless, there is still a need for optimization regarding their internalization to ensure an efficient drug delivery.

Keywords: Fe3O4; biocompatibility; immunotoxicology; nanoporous silica nanoparticles; phagocytosis; superparamagnetic iron oxide nanoparticles; targeted drug delivery.

Figure A1

Fluorescence microscopic images of macrophages after incubation for 5, 10, 20 and 90 min with the different MNPSNPs: 2 kDa mPEG-FITC, 5 kDa mPEG-FITC, and 10 kDa mPEG-FITC. Blue: nuclear staining with DAPI, Red: phalloidin stained F-actin of the cytoskeleton, Green: FITC labeled MNPSNPs, Objective: 63 × 1.4 with immersion oil.

Figure A1

Fluorescence microscopic images of macrophages after incubation for 5, 10, 20 and 90 min with the different MNPSNPs: 2 kDa mPEG-FITC, 5 kDa mPEG-FITC, and 10 kDa mPEG-FITC. Blue: nuclear staining with DAPI, Red: phalloidin stained F-actin of the cytoskeleton, Green: FITC labeled MNPSNPs, Objective: 63 × 1.4 with immersion oil.

Figure A2

Fluorescence microscopic images of macrophages after incubation for 90 min with 2 kDa mPEG-FITC MNPSNPs. Imaging of the XY-plane and the XZ-plane at the level of the dashed line. Blue: nuclear staining with DAPI, Red: phalloidin stained F-actin of the cytoskeleton, Green: FITC labeled MNPSNPs, Objective: 63 × 1.4 with immersion oil.

Figure 1

TEM images of unfunctionalized and functionalized MNPSNPs with 2 kDa mPEG-FITC, 5 kDa mPEG-FITC, and 10 kDa mPEG-FITC; FITC—Fluorescein-isothiocyanat Isomer I.

Figure 2

Nitrogen physisorption isotherms of unfunctionalized and functionalized MNPSNPs; empty squares: desorption branch, filled squares: adsorption branch.

Figure 3

Thermogravimetric curves of unfunctionalized and functionalized MNPSNPs.

Figure 4

Metabolic activity of macrophages and dendritic cells after incubation with different MNPSNPs for 24 and 48 h in comparison to the control. Data are shown as boxplot with min/max-whisker. The dotted line represents the control (10% aqua bidestillata; 100%); the dashed line represents the 70% viability limit according to ISO 10993-5;2009-06 [29]. One-way analysis of variance (ANOVA) with Bonferroni post-hoc test; * p ≤ 0.05, ** p ≤ 0.01 *** p ≤ 0.001; n = 6 each group; asterisks indicate significant differences versus control.

Figure 5

TNF-α concentration (pg/mL) in the supernatants of macrophages and dendritic cells after incubation with different MNPSNPs for 24 and 48 h. Data are shown as boxplot with min/max-whisker. Control equals 10% aqua bidestillata in the medium. One-way ANOVA with Bonferroni post-hoc test; * p ≤ 0.05, ** p ≤ 0.01; *** p ≤ 0.001; n = 6 each group; asterisks indicate significant differences versus control.

Figure 6

IL-6 concentration (pg/mL) in the supernatants of macrophages and dendritic cells after incubation with different MNPSNPs for 24 and 48 h. Data are shown as boxplot with min/max-whisker. Control equals 10% aqua bidestillata in the medium. One-way ANOVA with Bonferroni post-hoc test; * p ≤ 0.05; *** p ≤ 0.001; n = 6 each group; asterisks indicate significant differences versus control.

Figure 7

(A,B) Reactive oxygen species in macrophages (A, n = 6 each group) and dendritic cells (B, n = 4 each group) after incubation with different MNPSNPs over 24 h in comparison to the control (10% aqua bidestillata; dotted line, 100%). (C), Migrated dendritic cells (n = 6 each group) in relation to the control (10% aqua bidestillata; dotted line, 100%) after incubation with different MNPSNPs. (D) Colony forming units of E. coli in the cell lysate of MNPSNP-treated macrophages (n = 6 each group). Control equals 10% aqua bidestillata in the medium. (E) Corrected total intracellular fluorescence of the different MNPSNPs in 6–10 macrophages after incubation for 5-, 10-, 20-, and 90-min. Data are shown as boxplot with min/max-whisker. (A–D) One-way ANOVA with Bonferroni post-hoc test; * p ≤ 0.05, ** p ≤ 0.01; *** p ≤ 0.001; asterisks indicate significant differences versus control.

Figure 7

(A,B) Reactive oxygen species in macrophages (A, n = 6 each group) and dendritic cells (B, n = 4 each group) after incubation with different MNPSNPs over 24 h in comparison to the control (10% aqua bidestillata; dotted line, 100%). (C), Migrated dendritic cells (n = 6 each group) in relation to the control (10% aqua bidestillata; dotted line, 100%) after incubation with different MNPSNPs. (D) Colony forming units of E. coli in the cell lysate of MNPSNP-treated macrophages (n = 6 each group). Control equals 10% aqua bidestillata in the medium. (E) Corrected total intracellular fluorescence of the different MNPSNPs in 6–10 macrophages after incubation for 5-, 10-, 20-, and 90-min. Data are shown as boxplot with min/max-whisker. (A–D) One-way ANOVA with Bonferroni post-hoc test; * p ≤ 0.05, ** p ≤ 0.01; *** p ≤ 0.001; asterisks indicate significant differences versus control.