Iron(iii) chelated paramagnetic polymeric nanoparticle formulation as a next-generation T 1-weighted MRI contrast agent

Abstract

Magnetic resonance imaging (MRI) is a routinely used imaging technique in medical diagnostics. To enhance the quality of MR images, contrast agents (CAs) are used, which account for nearly 40% of MRI exams in the clinic globally. The most used CAs are gadolinium-based CAs (GBCAs) but the use of GBCAs has been linked with metal-deposition in vital organs. Gadolinium deposition has been shown to be correlated with nephrogenic systemic fibrosis, a fibrosis of the skin and internal organs. Therefore, there is an unmet need for a new CA alternative to GBCAs for T ₁-weighted Ce-MRI. Herein, we designed paramagnetic ferric iron(iii) ion-chelated poly(lactic-co-glycolic)acid nanoparticle formulation and routinely examined their application in Ce-MRI using clinical and ultra-high-field MRI scanners. Nanoparticles were monodispersed and highly stable at physiological pH over time with the hydrodynamic size of 130 ± 12 nm and polydispersity index of 0.231 ± 0.026. The T ₁-contrast efficacy of the nanoparticles was compared with commercial agent gadopentetate dimeglumine, called Magnevist®, in aqueous phantoms in vitro and then validated in vivo by visualizing an angiographic map in a clinical MRI scanner. Relaxivities of the nanoparticles in an aqueous environment were r ₁ = 10.59 ± 0.32 mmol^-1 s^-1 and r ₁ = 3.02 ± 0.14 mmol^-1 s^-1 at 3.0 T and 14.1 T measured at room temperature and pH 7.4, respectively. The clinically relevant magnetic field relaxivity is three times higher compared to the Magnevist®, a clinical GBCA, signifying its potential applicability in clinical settings. Moreover, iron is an endogenous metal with known metabolic safety, and the polymer and phospholipids used in the nanoconstruct are biodegradable and biocompatible components. These properties further put the proposed T ₁ agent in a promising position in contrast-enhanced MRI of patients with any disease conditions.

Fig. 1. Physiochemical characterization of iron(iii) chelated polymeric NPs. (A) Transmission Emission Micrograph (TEM) of NPs, (B) EDS spectroscopy showing the Fe content in the NPs, (C) hydrodynamic size of NPs as prepared and after 20 day incubation, and (D) zeta potential of PLGA NPs and Fe–PLGA NPs dispersed in PBS.

Fig. 2. Stability and loading/release study of iron(iii) chelated polymeric NPs. (A) Stability of NPs over 4 weeks period when stored at 4 °C showing the change of size and polydispersity index, (B) serum stability test to measure dynamic aggregation of PLGA NPs and Fe–PLGA NPs using 90% fetal bovine serum environment to find the rapid increase in optical density of 560 nm due to NPs aggregation by forming protein corona, (C) iron loading efficiency with the different initial feeding concentration of Fe³⁺ per mg PLGA, and (D) a comparative Fe and Gd release study from Fe–PLGA NPs and Magnevist® in simulated body fluid (SBF), respectively. Data represents mean ± S.D, n = 3.

Fig. 3. Oxidation of ascorbic acid catalyzed by Fe³⁺ ions. FeCl₃ catalyzed the oxidation reaction of ascorbic acid as shown by the decrease in absorbance at 265 nm whereas, Fe–PLGA NPs did not catalyze the reaction.

Fig. 4. In vitro biocompatibility and cellular uptake study. (A) Concentration-dependent cytotoxicity of NPs in MCF-7 cells with 48 h incubation, (B) representative confocal images showing comparative cellular uptake of NPs after 0.5, 1.5, and 3 h incubations, and (C) fluorescent quantification of cellular uptake by measuring corrected total cell fluorescent intensity of the cell population for each treatment and group. The data were statistically analyzes using one-way ANOVA with the Mann–Whitney test. (n = ∼50 cells, mean ± sd). ns = not significant, *p-value < 0.02, **p-value < 0.0045, ***p-value < 0.0001.

Fig. 5. Relaxivity study of iron(iii) chelated polymeric NPs in the low and ultra-high magnetic fields. (A and B) T₁ recovery curve of NPs at low field (3 T) and ultra-high-field (14.1 T) as a function of iron concentration, (C) T₁-weighted MR phantom images of iron(iii) chelated PLGA NPs aqueous suspensions with different concentrations corresponding to recovery curves (A) and (B), and (D) T₁ longitudinal relaxation rate against Fe³⁺ concentration measured at 3 T and 14.1 T MRI system at room temperature. Data represents mean ± S.D, n = 3.

Fig. 6. In vivo time-dependent T₁-weighted magnetic resonance image of mice at 3 T. (A) A representative 3D reconstruction of images acquired at injection using maximum intensity projection with pre-injection, at-injection, and post-injection after 10 min. (B) Representative 3D reconstruction images at 1, 2, and 3 h post-injection, respectively. (C) Representative 3D reconstruction of images acquired at injection, 15 min, and 1 h post injected images of Magnevist® (equivalent Gd concentration of 0.03 mmol kg⁻¹) from literature (ref. 49). The mice were intravenously injected with Fe–PLGA NPs (equivalent Fe concentration of 0.02 mmol kg⁻¹).