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Methods 1) P22: The P22 protein cage (60 nm) is bioengineered with an internal polymer network with amine functional groups allowing incorporation of ~9100 Gd-DTPA molecules per cage via the amine groups (Figure 1: [1]). This provides a per cage relaxivity of 70000 mMs, superior to Gd-DTPA for the equivalent Gd concentration. 2) Atherosclerosis Models: Both ApoE-deficient (ApoE) and FVB mice were used. ApoE mice develop atherosclerosis enhanced by high-fat diet. FVB mice develop macrophage-rich carotid lesions with carotid ligation in combination with high-fat diet and diabetes induction [2]. 3) P22-polymer-Gd in vivo MR imaging: Mice were injected intravenously with P22-polymer-Gd (N=5, 20 μmol Gd/kg, one-fifth the typical clinical dose) or Magnevist (N=1, 20 μmol Gd/kg). Vascular MRA at 1T was performed (Aspect M2, 500 mT/m, 2500 T/m/s) using 3D-SPGR (TR/TE=12 ms/2.1 ms, slice thickness=1 mm, FOV=5 cm, matrix=128x128, FA=45). Vessel wall MRI at 3T was performed (Signa HDx, GE Healthcare, 50mT/m, 150 T/m/s) with a phased-array mouse coil (RAPID MR International), using a double inversion recovery fast spin echo sequence (TR/TE= 400 ms/15 ms, slice thickness=1mm, FOV=3 cm, matrix= 256x256) up to 24 hours after injection. 4) RGD-targeted P22 ex vivo fluorescence imaging: Molecular targeting of P22 was evaluated by attaching RGD peptides externally, which targets the aVb3 integrin, upregulated on activated macrophages. ApoE mice (N=4) were injected intravenously with RGDP22 or RGD P22 (labeled with Cy5.5, 4 nmol/mouse). Forty-eight hours later, ex vivo fluorescence imaging was performed using Maestro imaging system (Cri, Woburn, MA). Maximum plaque signal intensities were measured and compared.

High-Gd-Payload P22 protein cage nanoparticles for imaging vascular inflammation