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The present work is focused on testing enzyme-based agents for the partial dissolution of calcium pyrophosphate (CaPP i ) deposits in the cartilages and synovial fluid of patients with pyrophosphate arthropathy (CPPD disease). Previously, we suggested that inorganic pyrophosphatases (PPases) immobilized on nanodiamonds of detonation synthesis (NDs) could be appropriate for this purpose. We synthesized and characterized conjugates of NDs and PPases from Escherichia coli and Mycobacterium tuberculosis. The conjugates showed high enzymatic activity and resistance to inhibition by calcium and fluoride. Here, we tested the effectiveness of pyrophosphate (PP i ) hydrolysis by the conjugates in an in vitro model system simulating the ionic composition of the synovial fluid and in the samples of synovial fluid of patients with CPPD via NMR spectroscopy. The conjugates of both PPases efficiently hydrolyzed triclinic crystalline calcium pyrophosphate (t-CPPD) in the model system. We evaluated the number of phosphorus-containing compounds in the synovial fluid, showed the possibility of PP i detection in it, and estimated the hydrolytic activity of the PPase conjugates. The soluble and immobilized PPases were able to hydrolyze a significant amount of PP i (1 mM) in the synovial fluid in short periods of time (24 h). The maximum activity was demonstrated for Mt-PPase immobilized on ND−NH−(CH 2 ) 6 −NH 2 (2.24 U mg −1 ).

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Pyrophosphate arthropathy is the mineralization defect in humans caused by the deposition of microcrystals of calcium pyrophosphate dihydrate in joint tissues. As a potential therapeutic strategy for the treatment of pyrophosphate arthropathy, delivery of exogenous pyrophosphate-hydrolyzing enzymes, inorganic pyrophosphatases (PPases), to the synovial fluid has been suggested. Previously, we synthesized the conjugates of Escherichia coli PPase (Ec-PPase) with detonation synthesis nanodiamonds (NDs) as a delivery platform, obtaining the hybrid biomaterial retaining high pyrophosphate-hydrolyzing activity in vitro. However, most known PPases including Ec-PPase in the soluble form are strongly inhibited by Ca 2+ ions. Because synovial fluid contains up to millimolar concentrations of soluble calcium, this inhibition might limit the in vivo application of Ec-PPasebased material in joint tissues. In this work, we proposed other bacterial PPases from Mycobacterium tuberculosis (Mt-PPase), which are resistant to the inhibition by Ca 2+ ions, as an active PP i-hydrolyzing agent. We synthesized conjugates of Mt-PPase with NDs and tested their activity under various conditions. Unexpectedly, conjugates of both Ec-PPase and Mt-PPase with aminated NDs retained significant hydrolytic activity in the presence of well-known mechanismbased PPase inhibitors, fluoride or calcium. The incomplete inhibition of PPases by fluoride or calcium was found for the first time.

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