Targeting angiogenesis using a C-type atrial natriuretic factor-conjugated nanoprobe and PET

Abstract

Sensitive, specific, and noninvasive detection of angiogenesis would be helpful in discovering new strategies for the treatment of cardiovascular diseases. Recently, we reported the (64)Cu-labeled C-type atrial natriuretic factor (CANF) fragment for detecting the upregulation of natriuretic peptide clearance receptor (NPR-C) with PET on atherosclerosis-like lesions in an animal model. However, it is unknown whether NPR-C is present and overexpressed during angiogenesis. The goal of this study was to develop a novel CANF-integrated nanoprobe to prove the presence of NPR-C and offer sensitive detection with PET during development of angiogenesis in mouse hind limb.

Methods: We prepared a multifunctional, core-shell nanoparticle consisting of DOTA chelators attached to a poly(methyl methacrylate) core and CANF-targeting moieties attached to poly(ethylene glycol) chain ends in the shell of the nanoparticle. Labeling of this nanoparticle with (64)Cu yielded a high-specific-activity nanoprobe for PET imaging NPR-C receptor in a mouse model of hind limb ischemia-induced angiogenesis. Histology and immunohistochemistry were performed to assess angiogenesis development and NPR-C localization.

Results: (15)O-H(2)O imaging showed blood flow restoration in the previously ischemic hind limb, consistent with the development of angiogenesis. The targeted DOTA-CANF-comb nanoprobe showed optimized pharmacokinetics and biodistribution. PET imaging demonstrated significantly higher tracer accumulation for the targeted DOTA-CANF-comb nanoprobe than for either the CANF peptide tracer or the nontargeted control nanoprobe (P < 0.05, both). Immunohistochemistry confirmed NPR-C upregulation in the angiogenic lesion with colocalization in both endothelial and smooth muscle cells. PET and immunohistochemistry competitive receptor blocking verified the specificity of the targeted nanoprobe to NPR-C receptor.

Conclusion: As evidence of its translational potential, this customized DOTA-CANF-comb nanoprobe demonstrated superiority over the CANF peptide alone for imaging NPR-C receptor in angiogenesis.

Conflict of interest statement

No other potential conflict of interest relevant to this article was reported.

Figures

FIGURE 1

Biodistribution of 64Cu-DOTA-CANF, 64Cu-DOTA-comb, and 64Cu-DOTA-CANF-comb in C57BL/6 mice (n = 3–4/group). (A) 64Cu-DOTA-CANF showing fast renal clearance and low blood retention. (B) Nontargeted 64Cu-DOTA-comb nanoparticle showing improved blood retention but high liver and spleen uptake. (C) 64Cu-DOTA-CANF-comb nanoprobe showing superior pharmacokinetics relative to both CANF peptide tracer alone and nontargeted control nanoprobe

FIGURE 2

PET/CT image of 64Cu-DOTA-CANF in HLI-induced angiogenesis model. (A) Coronal slice showing accumulation of 64Cu-DOTA-CANF tracer at injured limb on day 7. (B) Uptake of 64Cu-DOTA-CANF at ischemic (n = 6) and nonischemic (n = 6) limbs, as well as blocking study (n = 4). (C) Significant reduction of uptake upon competitive receptor blocking with coadministration of unlabeled CANF peptide. (D) Ischemic-to-nonischemic uptake ratios of nonblocking and blocking studies (n = 4, both).

FIGURE 3

PET/CT images of 64Cu-DOTA-CANF-comb and 64Cu-DOTA-comb in HLI-induced angiogenesis model 7 d after ischemia. (A) 64Cu-DOTA-CANF-comb in HLI model showing accumulation of activity in ischemic limb, with little observed in contralateral nonischemic limb. (B) 64Cu-DOTA-comb in HLI model showing weak uptake in both ischemic and nonischemic limbs. (C) Uptake of 64Cu-DOTA-CANF-comb (n = 8) and 64Cu-DOTA-comb (n = 7). (D) Ischemic-to-nonischemic uptake ratios of 64Cu-DOTA-CANF-comb (n = 8) and 64Cu-DOTA-comb (n = 7).

FIGURE 4

Immunofluorescent colocalization of NPR-C with neovessel endothelial cells and vascular smooth muscle cells in previously ischemic thigh muscle collected 7 d after femoral arterial surgery. (A, C, E, and G) Fluorescent and light images of previously ischemic hind limb tissue. (B, D, F, and H) Fluorescent and light images of contralateral nonischemic hind limb tissue. Coregistration (A, C, and D) (orange) of fluorescent images for PECAM-1 (green) or α-actin (green) to NPR-C (red). (E) Hematoxylin and eosin staining showing band of neovessels cut in longitudinal section corresponding to location of fluorescent staining for endothelium and NPR-C in A. (G) Hematoxylin and eosin staining showing coagulation necrosis of muscle and blue-stained nuclei of neovessel (center) and in previously ischemic tissue corresponding to location of fluorescent staining for smooth muscle cell and NPR-C (C). Interestingly, neovessels in previously ischemic tissue and existing capillaries in nonischemic tissue both stained for α-actin and NPR-C, although staining was much fainter in nonischemic tissue (D).