Charge-Modulated Synthesis of Highly Stable Iron Oxide Nanoparticles for In Vitro and In Vivo Toxicity Evaluation

Abstract

The surface charge of iron oxide nanoparticles (IONPs) plays a critical role in the interactions between nanoparticles and biological components, which significantly affects their toxicity in vitro and in vivo. In this study, we synthesized three differently charged IONPs (negative, neutral, and positive) based on catechol-derived dopamine, polyethylene glycol, carboxylic acid, and amine groups, via reversible addition-fragmentation chain transfer-mediated polymerization (RAFT polymerization) and ligand exchange. The zeta potentials of the negative, neutral, and positive IONPs were -39, -0.6, and +32 mV, respectively, and all three IONPs showed long-term colloidal stability for three months in an aqueous solution without agglomeration. The cytotoxicity of the IONPs was studied by analyzing cell viability and morphological alteration in three human cell lines, A549, Huh-7, and SH-SY5Y. Neither IONP caused significant cellular damage in any of the three cell lines. Furthermore, the IONPs showed no acute toxicity in BALB/c mice, in hematological and histological analyses. These results indicate that our charged IONPs, having high colloidal stability and biocompatibility, are viable for bio-applications.

Keywords: PEG ligands; biocompatibility; colloidal stability PEG ligands; iron oxide nanoparticles; toxicity.

Figure 1

RAFT polymerization reaction for synthesis of different charged polymer ligands. (a) (−) ligand, (b) (n) ligand, and (c) (+) ligand.

Figure 2

RAFT polymerization of (n) ligand showing (a,b) controllable polymer DP as a [Monomer] to [RAFT] ratio. GPC of (n) ligand in THF, showing a low PDI with a [Monomer]:[RAFT] ratio of 20:1 and [AIBN]:[RAFT] ratio of 1:1 (red line), and poor PDI without RAFT agent (black line).

Figure 3

Characterization of IONPs after ligand exchange. (a) Schematic of ligand exchange of OAc-IONPs with charged ligands, (b) TEM images of OAc-IONPs dispersed in hexane and three charged IONPs dispersed in DIW. (c) HD of OAc-IONPs and the three charged IONPs, and (d) zeta-potential of the three charge IONPs.

Figure 4

Colloidal stability of charged IONPs in DIW for three months and in cell culture media for two days. (a) HD and (b) zeta-potential of three differently charged IONPs in DIW for three months. (c) HD of three differently charged IONPs in RPMI 1640 medium and (d) in DMEM for two days.

Figure 5

(a) Effects of the three charged IONPs on the viabilities of three different cell lines, A549, Huh-7, and SH-SY5Y. All cells were exposed for 24 h to increasing concentrations upon 500 µg Fe/mL. Cell viability was analyzed by MTT assay. * p ≤ 0.05 and ** p ≤ 0.01 compared to the controls (Dunnett test with a one-way ANOVA). (b) Changes in cell morphology after treatment with 10 and 100 μg Fe/mL of the three charged IONPs by microscopy.

Figure 6

Blood chemistry of mice following injection of three charged IONPs at 2 µg/g and 10 µg/g for 24 h. (n = 3) Six parameters were analyzed. BUN and CREA were related to the kidney function, and AST, ALT, ALP, and TBIL were related to the liver function.