Biodegradable dendritic positron-emitting nanoprobes for the noninvasive imaging of angiogenesis

Abstract

A biodegradable positron-emitting dendritic nanoprobe targeted at alpha(v)beta(3) integrin, a biological marker known to modulate angiogenesis, was developed for the noninvasive imaging of angiogenesis. The nanoprobe has a modular multivalent core-shell architecture consisting of a biodegradable heterobifunctional dendritic core chemoselectively functionalized with heterobifunctional polyethylene oxide (PEO) chains that form a protective shell, which imparts biological stealth and dictates the pharmacokinetics. Each of the 8 branches of the dendritic core was functionalized for labeling with radiohalogens. Placement of radioactive moieties at the core was designed to prevent in vivo dehalogenation, a potential problem for radiohalogens in imaging and therapy. Targeting peptides of cyclic arginine-glycine-aspartic acid (RGD) motifs were installed at the terminal ends of the PEO chains to enhance their accessibility to alpha(v)beta(3) integrin receptors. This nanoscale design enabled a 50-fold enhancement of the binding affinity to alpha(v)beta(3) integrin receptors with respect to the monovalent RGD peptide alone, from 10.40 nM to 0.18 nM IC(50). Cell-based assays of the (125)I-labeled dendritic nanoprobes using alpha(v)beta(3)-positive cells showed a 6-fold increase in alpha(v)beta(3) receptor-mediated endocytosis of the targeted nanoprobe compared with the nontargeted nanoprobe, whereas alpha(v)beta(3)-negative cells showed no enhancement of cell uptake over time. In vivo biodistribution studies of (76)Br-labeled dendritic nanoprobes showed excellent bioavailability for the targeted and nontargeted nanoprobes. In vivo studies in a murine hindlimb ischemia model for angiogenesis revealed high specific accumulation of (76)Br-labeled dendritic nanoprobes targeted at alpha(v)beta(3) integrins in angiogenic muscles, allowing highly selective imaging of this critically important process.

Fig. 1.

Preparation of PET nanoprobes targeted at α_vβ₃ integrin.

Fig. 2.

Cell uptake studies. (A) Percentage total cell-associated fraction, cell-internalized fraction, and surface-bound fraction for the targeted nanoprobe in α_vβ₃-positive M21 cells. (B) Percentage cell-internalized fraction for the targeted nanoprobe in the absence and presence of the block and the nontargeted nanoprobe in α_vβ₃-positive M21 cells. All values are normalized to the protein content per well. Total cell-associated fraction represents the sum of cell-internalized fraction and surface-bound fraction. %ID/mg Protein refers to percentage injected or administered dose per milligram of protein.

Fig. 3.

Biodistribution of nontargeted (A) and targeted (B) nanoprobes in healthy rats. Bars represent the mean (n = 4) percentage injected dose per gram of tissue (%ID/gram) and standard deviations (*, P < 0.05 with respect to RGD dendritic nanoprobe).

Fig. 4.

Laser Doppler perfusion imaging in the hindlimb ischemia model. Images from a representative animal showing the perfusion of the hindlimbs immediately after surgery (Day 0) to induce ischemia of the right leg (shown on the left) and 1 week later. Blue and green areas reflect low levels of perfusion (0–200), whereas yellow, red, and white reflect higher flows (800–1,000). Flow ratios were calculated as the flow in the midthigh in the ischemic leg compared with the flow in the same area in the nonischemic leg. Relative perfusion increased significantly over 1 week after surgery (P < 0.008, day 0 vs. day 7).

Fig. 5.

Noninvasive PET/CT images of angiogenesis induced by hindlimb ischemia in a murine model. (A) Nontargeted dendritic nanoprobes (shown bottom center). (B) Uptake of α_vβ₃-targeted dendritic nanoprobes was higher in ischemic hindlimb (left side of image) as compared with control hindlimb (right side of image).

Fig. 6.

Ischemic to nonischemic hindlimb (R/L) ratios of targeted and nontargeted nanoprobes and representative photomicrographs of thigh muscle showing α_vβ₃ receptor staining 1 week after induction of hindlimb ischemia. (A and B) Ischemic to nonischemic hindlimb (R/L) ratios of targeted and nontargeted nanoprobes from imaging analysis (B) and γ-well counting of tissues. Bars represent the mean and standard deviation (*, P < 0.05). (C) Sections from the ischemic hindlimb (Upper) showing patches of staining for α_vβ₃ receptors around neovessels and from the contralateral nonischemic hindlimb (Lower) showing minimal staining for α_vβ₃ receptors.