Composition-Tunable Ultrasmall Manganese Ferrite Nanoparticles: Insights into their In Vivo T1 Contrast Efficacy

Abstract

The development of a highly efficient, low-toxicity, ultrasmall ferrite nanoparticle-based T₁ contrast agent for high-resolution magnetic resonance imaging (MRI) is highly desirable. However, the correlations between the chemical compositions, in vitro T₁ relaxivities, in vivo nano-bio interactions and toxicities remain unclear, which has been a challenge in optimizing the in vivo T₁ contrast efficacy. Methods: Ultrasmall (3 nm) manganese ferrite nanoparticles (Mn_xFe_3-xO₄) with different doping concentrations of the manganese ions (x = 0.32, 0.37, 0.75, 1, 1.23 and 1.57) were used as a model system to investigate the composition-dependence of the in vivo T₁ contrast efficacy. The efficacy of liver-specific contrast-enhanced MRI was assessed through systematic multiple factor analysis, which included the in vitro T₁ relaxivity, in vivo MRI contrast enhancement, pharmacokinetic profiles (blood half-life time, biodistribution) and biosafety evaluations (in vitro cytotoxicity testing, in vivo blood routine examination, in vivo blood biochemistry testing and H&E staining to examine the liver). Results: With increasing Mn doping, the T₁ relaxivities initially increased to their highest value of 10.35 mM^-1s^-1, which was obtained for Mn_0.75Fe_2.25O₄, and then the values decreased to 7.64 m M^-1s^-1, which was obtained for the Mn_1.57Fe_1.43O₄ nanoparticles. Nearly linear increases in the in vivo MRI signals (ΔSNR) and biodistributions (accumulation in the liver) of the Mn_xFe_3-xO₄ nanoparticles were observed for increasing levels of Mn doping. However, both the in vitro and in vivo biosafety evaluations suggested that Mn_xFe_3-xO₄ nanoparticles with high Mn-doping levels (x > 1) can induce significant toxicity. Conclusion: The systematic multiple factor assessment indicated that the Mn_xFe_3-xO₄ (x = 0.75-1) nanoparticles were the optimal T₁ contrast agents with higher in vivo efficacies for liver-specific MRI than those of the other compositions of the Mn_xFe_3-xO₄ nanoparticles. Our work provides insight into the optimization of ultrasmall ferrite nanoparticle-based T₁ contrast agents by tuning their compositions and promotes the translation of these ultrasmall ferrite nanoparticles for clinical use of high-performance contrast-enhanced MRI.

Keywords: MRI; composition effect; in vivo T1 contrast efficacy; ultrasmall ferrite nanoparticles.

Figure 1

(a-f) TEM images of ultrasmall Mn_xFe_3-xO₄ nanoparticles. The insets are the high-resolution TEM images. (g) XRD patterns of ultrasmall Mn_xFe_3-xO₄ nanoparticles. XPS spectra of ultrasmall Mn_xFe_3-xO₄ nanoparticles, (h) Fe 2p and (i) Mn 2p.

Figure 2

Magnetic characterization of 3 nm Mn_xFe_3-xO₄ nanoparticles. (a) Field-dependent magnetization curves (M-H) of ultrasmall Mn_xFe_3-xO₄ nanoparticles at 300 K. (b) The magnetizations at 3 T of the ultrasmall Mn_xFe_3-xO₄ samples.

Figure 3

Hydrodynamic size of (a) MnFe₂O₄@mPEG1000, (b) MnFe₂O₄@mPEG2000, and (c) MnFe₂O₄@mPEG5000 in water. The insets are digital photographs of the aqueous MnFe₂O₄ nanoparticles dispersions. (d)-(f) Time-dependent hydrodynamic size of ultrasmall MnFe₂O₄ nanoparticles modified with (d) mPEG1000, (e) mPEG2000 and (f) mPEG5000, respectively. (g) T₁-weighted phantom imaging of ultrasmall MnFe₂O₄ nanoparticles with different phosphorylated mPEG. (h) T₁ relaxation rate of the MnFe₂O₄ nanoparticles different PEG chain lengths at various [Fe+Mn] concentrations. (i) r₁ value and r₂/r₁ ratio of MnFe₂O₄ nanoparticles as a function of the mPEG molecular weight.

Figure 4

MR contrast effects of ultrasmall Mn_xFe_3-xO₄ nanoparticles upon changes in the Mn doping level. (a) T₁-weighted images of Mn_xFe_3-xO₄ nanoparticles. (b) T₁ relaxation rate of the Mn_xFe_3-xO₄ nanoparticles and (c) r₁ relaxivities and r₂/r₁ ratio of ultrasmall Mn_xFe_3-xO₄ nanoparticles.

Figure 5

In vivo MR imaging of UMFNPs. (a)T₁-weighted MR images of liver at 0, 3, 10, 30, 60, 90, 120, and 180 min after intravenous injection of 3 nm Mn_xFe_3-xO₄ nanoparticles and (b) quantification analysis of MR T₁ signals changes.

Figure 6

In vivo behaviors of ultrasmall Mn_xFe_3-xO₄ nanoparticles as determined by measuring Mn levels with ICP-MS. (a) Blood circulation of ultrasmall Mn_xFe_3-xO₄ nanoparticles. The pharmacokinetics of ultrasmall Mn_xFe_3-xO₄ nanoparticles followed the two-compartment model. (b) Correlation of elimination half-life and clearance of ultrasmall Mn_xFe_3-xO₄ nanoparticles. (c, d) Biodistribution of ultrasmall Mn_xFe_3-xO₄ nanoparticles in Balb/c mice at (c) 3 h and (d) 24 h post injection.

Figure 7

Relative viabilities of (a) Chang liver cells and (b) HepG2 cells incubated with ultrasmall Mn_xFe_3-xO₄ nanoparticles at various concentrations using standard cck-8 colorimetric assays. Error bars = SEM; * p < 0.05; ** p < 0.01; *** p < 0.001. p value is used to statistically analyze difference among groups at the [Fe+Mn] concentration of 50 µg/mL.

Figure 8

In vivo biosafety assessment of ultrasmall Mn_xFe_3-xO₄ nanoparticles. (a-h) Routine blood analysis: (a) red blood cells (RBC), (b) hemoglobin (HGB), (c) white blood cell (WBC), (d) hematocrit (HCT), (e) mean corpuscular hemoglobin (MCH), (f) mean corpuscular hemoglobin concentration (MCHC), (g) platelets (PLT) and (h) mean corpuscular volume (MCV); (i-o) Blood biochemistry test: (i) alanine transferase (ALT), (j) aspartate transferase (AST), (k) total protein (TP), (l) albumin (ALB), (m) total biliary acid (TBA), (n) direct bilirubin (DBIL) and (o) alkaline phosphatase (ALP).

Figure 9

H&E stained images of the liver of the mice harvested from control group and treated groups at 1 days after intravenous injection of ultrasmall Mn_xFe_3-xO₄ nanoparticles. Scale bar = 100 nm.

Figure 10

The comparison and correlation analysis of Mn concentration with in vitro T₁ relaxivity, in vivo nano-bio interactions and biosafety.