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

Background

The bacteriophage P22 protein cage can be bioengineered to contain a high-relaxivity gadolinium (Gd) payload internally and targeting ligands externally. It also enables phage-library-based identification of novel targets. Thus, P22 may have advantages for molecular/cellular imaging by MRI.

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 mM^-1s^-1, superior to Gd-DTPA for the equivalent Gd concentration.

Figure 1

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^TM, 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 αVβ3 integrin, upregulated on activated macrophages. ApoE^-/- mice (N=4) were injected intravenously with RGD⁺P22 or RGD^-P22 (labeled with Cy5.5, 4 nmol/mouse). Forty-eight hours later, ex vivo fluorescence imaging was performed using Maestro^TM imaging system (Cri, Woburn, MA). Maximum plaque signal intensities were measured and compared.

Results

Low dose P22-polymer-Gd showed strong enhancement for 1T vascular MRA (Figure 2). It also showed clear enhancement of the aortic wall (ApoE^-/-) and ligated carotid (FVB) at 3T (Figure 3). Ex vivo fluorescence imaging showed the accumulation of both RGD⁺P22 or RGD^-P22 in atherosclerotic lesions (Figure 4). RDG targeting enhanced plaque uptake (RGD⁺P22: 0.025 ± 0.002 counts/sec vs. RGD^-P22: 0.005 ± 0.004 counts/sec, p=0.05).

Figure 2

Figure 3

Figure 4

Conclusions

The P22 protein cage nanoparticle demonstrates both internal high-relaxivity Gd-loading for in vivo MRI as well as external RGD-targeting for enhanced uptake in vascular inflammation. Thus, P22 is a novel, multi-functional nanoparticle platform for targeted-imaging of atherosclerosis.

Funding

GE healthcare, Kowa, Inc.