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Synthesis of Inorganic Pyrophosphatase-Nanodiamond Conjugates Resistant to Calcium and Fluoride
Pyrophosphate arthropathy is the mineralization defect in humanscaused by the deposition of microcrystals of calcium pyrophosphate dihydrate injoint tissues. As a potential therapeutic strategy for the treatment of pyrophosphatearthropathy, delivery of exogenous pyrophosphate-hydrolyzing enzymes, inorganicpyrophosphatases (PPases), to the synovial fluid has been suggested. Previously,we synthesized the conjugates of Escherichia coli PPase (Ec-PPase) with detonationsynthesis nanodiamonds (NDs) as a delivery platform, obtaining the hybridbiomaterial retaining high pyrophosphate-hydrolyzing activity in vitro. However,most known PPases including Ec-PPase in the soluble form are strongly inhibitedby Ca2+ ions. Because synovial fluid contains up to millimolar concentrations ofsoluble calcium, this inhibition might limit the in vivo application of Ec-PPase-based material in joint tissues. In this work, we proposed other bacterial PPasesfrom Mycobacterium tuberculosis (Mt-PPase), which are resistant to the inhibition by Ca2+ ions, as an active PPi-hydrolyzing agent.We synthesized conjugates of Mt-PPase with NDs and tested their activity under various conditions. Unexpectedly, conjugates ofboth Ec-PPase and Mt-PPase with aminated NDs retained significant hydrolytic activity in the presence of well-known mechanism-based PPase inhibitors, fluoride or calcium. The incomplete inhibition of PPases by fluoride or calcium was found for the first time.