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

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 αvβ3 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 whole-body 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).

figure1

Figure 1

figure2

Figure 2

figure3

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.

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Correspondence to Toshiro Kitagawa.

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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Kitagawa, T., Kosuge, H., Uchida, M. et al. RGD targeting of human ferritin iron oxide nanoparticles can enhance in vivo carotid MRI of experimental atherosclerosis. J Cardiovasc Magn Reson 13, P373 (2011). https://doi.org/10.1186/1532-429X-13-S1-P373

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Keywords

  • Protein Cage
  • Lumen Size
  • Angiogenic Endothelial Cell
  • Human Ferritin
  • Carotid Lumen