Selection and identification of a novel bone-targeting peptide for biomedical imaging of bone

Abstract

The global burden of bone-related diseases is increasing in the aging society; thus, improved bone targeted imaging for their early identification and treatment are needed. In this study, we screened novel peptide ligands for hydroxyapatite, a major inorganic component of teeth and bones, and identified a peptide enabling in vivo bone targeting and real-time fluorescence bone detection. To isolate peptides highly specific for hydroxyapatite, we used negative and positive selection from a randomized 8-mer peptide phage library and identified hydroxyapatite-specific peptides (HA-pep2, HA-pep3, and HA-pep7). Among these three peptides, HA-pep3 showed the highest binding capacity and superior dissociation constant towards hydroxyapatite surfaces over time (~ 88.3% retained on hydroxyapatite after two weeks). Furthermore, HA-pep3 was highly specific for hydroxyapatite compared to other calcium salt-based materials. Using this superior specificity, HA-pep3 showed higher accumulation in skull, spine, and joints in comparison with scrambled control peptide during real-time whole-body imaging. Ex vivo analysis of the major organs and bone from mice demonstrated that the fluorescence intensity in bone was about 3.32 folds higher in the case of HA-pep3 than the one exhibited by the scrambled control peptide. Our study identified a novel approach for targeting ligands for bone specific imaging and can be useful for drug delivery applications.

Figure 1

Overview of the screening of hydroxyapatite specific peptides and in vivo bone-targeting imaging.

Figure 2

Phage selection of hydroxyapatite binding peptides from a randomized 8-mer peptide phage library. (a) Ratio of output/input phages after one, two, three, and four rounds of selection against hydroxyapatite. (b) The ratio of the output/input phage of hydroxyapatite-specific positive clones after the fourth biopanning.

Figure 3

Binding studies of FITC-labeled HA-specific peptides. (a) HA-binding test of selected peptides (HA-pep2, HA-pep3, HA-pep7), positive peptide(E7) and negative peptide. (b) Binding assay of HA-binding peptides in different concentrations and (c) incubation times.

Figure 4

Release kinetics of HA-binding peptides and positive peptide (E7) on hydroxyapatite.

Figure 5

The specificity of (a) the HA-pep3 and (b) acidic oligopeptide as positive peptide for the biologically relevant calcium salts. HA = hydroxyapatite, CC = calcium carbonate, CPP = calcium pyrophosphate, CP = calcium phosphate.

Figure 6

In vivo application of the HA-pep3. (a) Viability of Saos-2 cells treated with HA-pep3. (b) In vivo fluorescence imaging of BALB/c nude mice at 3 and 6 h after intravenous injection of Cy5.5-scramble peptide and Cy5.5-HA pep3. Skin, skull, spine, and joint are marked as dashed lines. (c) Ex vivo fluorescence imaging of bone and major tissues using the HA-pep3 and scramble peptide. (d) Fluorescence signal intensity graph of HA-pep3 and scramble peptide in bone and major tissues.