RGD targeting of human ferritin iron oxide nanoparticles can enhance in vivo carotid MRI of experimental atherosclerosis

Abstract

Background: Human ferritin (HFn) is a promising nanoscale protein cage platform for molecular/cellular imaging, and we have developed engineered HFn nanoparticles as MRI agents. Inflammation and angiogenesis contribute to atherosclerosis, and RGD is a well-studied ligand of the alphavbeta3 integrin expressed by activated macrophages and angiogenic endothelial cells. Purpose: To evaluate RGD-conjugated HFn-iron oxide nanoparticles for enhanced in vivo MRI detection of murine carotid atherosclerosis. Methods: 1) Mice Fourteen FVB mice underwent left carotid ligation after 4 weeks of high-fat diet and diabetes induction by streptozotocin. 2) RGD-conjugated HFn-iron oxide nanoparticles Using the recombinant human heavy-chain ferritin protein cage, HFn was genetically engineered to display 24 copies of an RGD-4C peptide (CDCRGDCFC) on the exterior surface of the protein cage. Magnetite (Fe3O4) was encapsulated in interior cavities of RGD-conjugated HFn (RGD+ HFn) and non-targeted HFn (RGD- HFn) at loading factors of 5000Fe per cage, giving R2 values of 93 mM-1s-1 (magnetite diameter: 5-7nm, exterior diameter: 12nm). The injected dose was adjusted to 25mgFe/kg. 3) MRI Two weeks post ligation, mice were imaged on a wholebody 3T MRI scanner (Signa HDx, GE Healthcare) with a phased array mouse coil (RAPID MR International), using a gradient echo sequence (TR/TE=100ms/10ms, slice thickness=1.0mm, FOV=3cm, matrix=256x256, FA=60, NEX=10). Mice were then injected with either RGD+ (n=7) or RGD- (n=7) HFn nanoparticles, followed by MRI at 24 and 48 hours post injection. The nanoparticle accumulation was assessed by measuring the extent of T2*-induced reduction in carotid lumen size (% reduction of carotid lumen area). 4) Histology Perl's iron staining was performed to identify accumulation of the nanoparticles in the carotid lesions. Results: Both RGD+ and RGD- HFn nanoparticles caused a reduction in lumen size of the ligated left carotid arteries at 48 hrs due to T2* signal loss (p<0.001 vs. preinjection, Figures 1, 2), but the effect was significantly greater with RGD+ HFn (p=0.01 vs. RGD- HFn). There was no significant lumen reduction in the non-ligated (control) right carotid arteries. Perl's iron staining confirmed greater accumulation of RGD+ HFn in the lesion compared to RGD- HFn, primarily in neointimal macrophages (Figure 3). Conclusions: Human ferritin protein cage is a versatile nanoparticle imaging platform for in vivo cellular/molecular MRI, with enhanced atherosclerosis imaging through multivalent RGD targeting(Figure Presented).