Biological evaluation of Fe₃O₄-poly(L-lactide)-poly(ethylene glycol)-poly(L-lactide) magnetic microspheres prepared in supercritical CO₂.

The biocompatibility of Fe₃O₄-poly(L-lactide)-poly(ethylene glycol)-poly(L-lactide) magnetic microspheres (Fe₃O₄-PLLA-PEG-PLLA MMPs) prepared in a process of suspension-enhanced dispersion by supercritical CO₂ (SpEDS) was evaluated at various levels: cellular, molecular, and integrated. At the cellular level, the investigations of cytotoxicity and intracellular reactive oxygen species (ROS) generation indicate that the polymer-coated MMPs (2.0 mg/mL) had a higher toxicity than uncoated Fe₃O₄ nanoparticles, which led to about 20% loss of cell viability and an increase (0.2 fold) in ROS generation; the differences were not statistically significant (p > 0.05). However, an opposite phenomenon was observed in tests of hemolysis, which showed that the MMPs displayed the weakest hemolytic activity, namely only about 6% at the highest concentration (20 mg/mL). This phenomenon reveals that polymer-coated MMPs created less toxicity in red blood cells than uncoated Fe₃O₄ nanoparticles. At the molecular level, the MMPs were shown to be less genotoxic than Fe₃O₄ nanoparticles by measuring the micronucleus (MN) frequency in CHO-K1 cells. Furthermore, the mRNA expression of pro-inflammatory cytokines demonstrates that polymer-coated MMPs elicited a less intense secretion of pro-inflammatory cytokines than uncoated Fe₃O₄ nanoparticles. Acute toxicity tests of MMPs show quite a low toxicity, with an LD₅₀ > 1575.00 mg/kg. The evidence of low toxicity presented in the results indicates that the Fe₃O₄-PLLA-PEG-PLLA MMPs from the SpEDS process have great potential for use in biomedical applications.