PET/CT Imaging of Chemokine Receptors in Inflammatory Atherosclerosis Using Targeted Nanoparticles

Abstract

Atherosclerosis is inherently an inflammatory process that is strongly affected by the chemokine-chemokine receptor axes regulating the trafficking of inflammatory cells at all stages of the disease. Of the chemokine receptor family, some specifically upregulated on macrophages play a critical role in plaque development and may have the potential to track plaque progression. However, the diagnostic potential of these chemokine receptors has not been fully realized. On the basis of our previous work using a broad-spectrum peptide antagonist imaging 8 chemokine receptors together, the purpose of this study was to develop a targeted nanoparticle for sensitive and specific detection of these chemokine receptors in both a mouse vascular injury model and a spontaneously developed mouse atherosclerosis model.

Methods: The viral macrophage inflammatory protein-II (vMIP-II) was conjugated to a biocompatible poly(methyl methacrylate)-core/polyethylene glycol-shell amphiphilic comblike nanoparticle through controlled conjugation and polymerization before radiolabeling with (64)Cu for PET imaging in an apolipoprotein E-deficient (ApoE(-/-)) mouse vascular injury model and a spontaneous ApoE(-/-) mouse atherosclerosis model. Histology, immunohistochemistry, and real-time reverse transcription polymerase chain reaction were performed to assess the plaque progression and upregulation of chemokine receptors.

Results: The chemokine receptor-targeted (64)Cu-vMIP-II-comb showed extended blood retention and improved biodistribution. PET imaging showed specific tracer accumulation at plaques in ApoE(-/-) mice, confirmed by competitive receptor blocking studies and assessment in wild-type mice. Histopathologic characterization showed the progression of plaque including size and macrophage population, corresponding to the elevated concentration of chemokine receptors and more importantly increased PET signals.

Conclusion: This work provides a useful nanoplatform for sensitive and specific detection of chemokine receptors to assess plaque progression in mouse atherosclerosis models.

Keywords: PET/CT; atherosclerosis; chemokine receptor; nanoparticle; viral macrophage inflammatory protein-II.

FIGURE 1

Synthesis of vMIP-II-Comb (α- bromide, vMIP-II)

FIGURE 2

Biodistribution of ⁶⁴Cu-vMIP-II-Comb in WT C57BL/6 mice (n=4/group).

FIGURE 3

(A) Representative 24 h images of ⁶⁴Cu-vMIP-II-Comb, blocking, and ⁶⁴Cu-Comb in injured ApoE^-/- mouse. (B) Quantitative uptake analysis. *** P < 0.001, n=6/group.

FIGURE 4

(A) Representative 24 h PET/CT images and (B) uptake analysis of ⁶⁴Cu-vMIP-II-Comb, blocking, and ⁶⁴Cu-Comb in ApoE^-/- mouse spontaneous atherosclerosis model. *** P < 0.001, n=6/group.

FIGURE 5

Representative (A) Hematoxylin and eosin staining (40×) and (B) F4/80 staining (40×) of plaques at 20 and 37 weeks on HCD in the ApoE^-/- mouse spontaneous atherosclerosis model and (C) Measurement of plaque area and F4/80 (brown) positive area at two time points (*P < 0.05, ** P < 0.005, n=3/group).

FIGURE 6

RT-PCR analysis of 8 chemokine receptors in (A) injured and sham-operated femoral arteries at 2 weeks post injury in ApoE^-/- mice; WT mice fed with normal chow and ApoE^-\- mice fed with HCD at (B) 20 and (C) 37 weeks (n=3-5/group).