Inorganic Pyrophosphatase-Nanodiamond Conjugates Hydrolyze Pyrophosphate in Human Synovial Fluid

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-(CH₂)₆-NH₂ (2.24 U mg^-1).