Life science
Medicine / Healthcare, Drug development

An acid-activatable fluorescent probe for imaging osteocytic bone
resorption activity in deep bone cavities
Department of Applied Chemistry, Graduate School of Engineering
Professor Kazuya Kikuchi

Researchmap https://researchmap.jp/kikuchilab315

Assistant Professor Masafumi Minoshima

Researchmap https://researchmap.jp/cbatgcm_m33

Life science

Abstractrole in remodeling bone-matrix in health and disease.

A pH-activatable fluorescent probe (pHocas-RIS) with moderatebone-binding affinity was developed by conjugating a BODIPY
fluorophore to bisphosphonate-targeting risedronate ligands. The
probe enabled the imaging of acidic osteocytic lacunae that contain bone resorbing osteocytes in deep bone tissues in living animals.

▍ Background & Results
Long lived osteocytes constitute around 90–95% of all bonecells, which exist within disk-shaped cavities called osteocytic
lacunae that are present throughout the mineralized bone matrix.
Osteocytes are known to directly remodel the bone walls by lacunae bone wall mineralisation producing larger cavities in a process
known as osteocytic osteolysis. However, the physiological role ofosteocytes in direct bone mineralisation is still open to debate, sothe availability of high-resolution real time imaging method would
be extremely useful.
Thus, we developed a new probe (pHocas-RIS) that could beused to visualize the low pH environments of osteocytic lacunae
that contained acid secreting osteocytes. Conjugation of the moderate bone binding drug risedronate to a pH-activatable BODIPY
fluorophore afforded pHocas-RIS in order to penetrate osteocytic
lacunae cavities embedded deep within the bone matrix. The fluorescence intensity of the pHocas-RIS probe increased approximately 15-fold as the pH decreased from 8.0 to 4.0, with changes
in its quantum yield at different pH levels used to calculate the pKavalue of its aniline moiety as 6.8.
After subcutaneous administration of the probe to mice over a
3 day period followed by aesthetisation, we performed two-photon imaging of the medullary cavities of their thin calvaria parietal
bones. Imaging studies revealed the presence of small numbersof ring-shaped green fluorescence signals of the protonated pHocas-RIS that were present in bone obtained from deep corticalregions. It is proposed that these fluorescent signals originate fromprobes bound to the walls of osteocytic lacunae that containedbone resorptive osteocytes that were actively secreting acid tocreate a low pH environment. The pH responsiveness of the pHocas-RIS probe within the lacunae of the bone matrix was confirmed
by carrying out ex vivo fluorescent imaging of cryogenically prepared deep bone slices at different pH levels.

▍ Significance of the research and Future perspective
These results clearly demonstrated that the intensity of thepH-activatable fluorescent response of the pHocas-RIS probe canbe used to effectively image the pH of osteocytic lacunae in livingbone. Our pH responsive probe can be used to visualize the bonemineralizing activities of acid producing osteocytes in real time,thus providing a valuable imaging method to explore their central

P a t e n t
Hashimoto, Ryu; Minoshima, Masafumi; Kikuta, Junichi et al. An Acid Activatable Fluorescence Probe for Imaging Osteocytic Bone Resorption Activity in Deep Bone Cavities. Angew. Chem. Int. Ed.
2020; 59, 20996-21000. doi: 10.1002/anie.202006388.
Kowada, Toshiyuki; Kikuta, Junichi; Kubo, Atsuko et al. In Vivo Fluorescence Imaging of Bone-resorbing Osteoclasts. J. Am. Chem. Soc. 2011; 133 (44), 17772–17776. doi: 10.1021/ja2064582.
Tr e a t i s e
Sano, Hiroshige; Kikuta, Junichi; Furuya, Masayuki et al. Intravital Bone Imaging by Two-photon Excitation Microscopy to Identify Osteocytic Osteolysis In Vivo, Bone, 2015; 74, 134–139. doi: 10.1016/j.bone.2015.01.013.
Maeda, Hiroki; Kowada, Toshiyuki; Kikuta, Junichi et al. Real-time Intravital Imaging of pH Variation Associated with Osteoclast Activity Nat. Chem. Biol. 2016; 12, 597–585. doi: 10.1038/nchembio.2096.
URLK e y w o r d fluorescent probe, osteocytes, intravital imaging, bone resorption, pH