Nanodiamond (ND) particles are popular platforms for the immobilization of molecular species. In the present research, enzyme Escherichia coli inorganic pyrophosphatase (PPase) was immobilized on detonation ND through covalent or noncovalent bonding and its enzymatic activity was characterized. Factors affecting adsorption of PPase such as ND size and surface chemistry were studied. The obtained material is a submicron size association of ND particles and protein molecules in approximately equal amounts. Both covalently and noncovalently immobilized PPase retains a significant enzymatic activity (up to 95% of its soluble form) as well as thermostability. The obtained hybrid material has a very high enzyme loading capacity (∼1 mg mg(-1)) and may be considered as a promising delivery system of biologically active proteinaceous substances, particularly in the treatment of diseases such as calcium pyrophosphate crystal deposition disease and related pathologies. They can also be used as recoverable heterogeneous catalysts in the traditional uses of PPase.

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 ).

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.

Background:Calcium pyrophosphate deposition (CPPD) disease, alternatively known as pseudogout, is a type of arthropathy caused by a deposition of microcrystals of calcium pyrophosphate (CaPPi) in joint tissues, particularly in fibrocartilage and hyaline cartilage. Up to date, specific treatment of CPPD disease has not been developed. Conventional therapy is focused on anti-inflammatory and immunosuppressive treatment aimed at the prevention of acute symptoms. Previously we suggested for this purpose inorganic pyrophosphatases (PPases) immobilized on nanodiamonds of detonation synthesis (NDs) [1]. We synthesized and characterized conjugates of NDs with PPases fromEscherichia coliandMycobacterium tuberculosis(Ec-PPase or Mt-PPase) using amino-substituted NDs with (ND-L) or without linker (ND-NH2). The conjugates showed high enzymatic activity in thein vitromodel system simulating the ionic composition of synovial fluidObjectives:The present work is focused on testing enzyme-based agents for the partial dissolution of calcium pyrophosphate deposits in the synovial fluid of patients with CPPD disease.Methods:PPase activity of soluble and immobilized PPases was evaluated as the rate of PPi decrease with time. 1 mM PPiwas added to the synovial fluid of patients with CPPD containing the samples of PPases, and the amount of PPiin the samples was determined every 2-4 hours using31P NMR spectroscopy.Results:We demonstrated the possibility of differential detection of pyrophosphate (PPi) among the spectrum of phosphorus-containing compounds in the synovial fluid and used the rate of decrease of exogenous PPito estimate hydrolytic activity of PPases and PPase-based conjugates. The hydrolysis followed the first order reaction kinetics and the rate constants by different samples of PPases are presented in Table 1. Non-enzymatic hydrolysis of exogenous PPiwas quite moderate or not observed at all. Ec-PPase (both soluble and immobilized) showed very low hydrolytic activity with the residual PPilevels above 80%. However, all samples of Mt-PPase showed significant hydrolytic activity. In two cases of three, PPiwas almost completely hydrolyzed within 20 hours. The maximum activity (2.24 U•mg-1) was shown by Mt-PPase immobilized on ND-L. Hydrolytic activity for all studied enzymes is only 1-2% of such values under optimalin vitroconditions, most probably due to the inhibiting effect of calcium in synovial fluid. Nevertheless, the activity of Mt-PPase-based samples was comparable with the values typical for some classes of hydrolases. It allows us to consider the suggested PPase-based materials as promising agents for the hydrolysis of PPiin the joint tissuesin vivo.Table 1.PPihydrolysis in the synovial fluid by soluble or immobilized PPases.Samplesk, hr-1A, U·mg-1Control-0.010 ± 0.0010ND-NH200ND-L0.013 ± 0.0020Ес-РРaseSoluble0.025 ± 0.0030.33 ± 0.04Immobilized on ND-NH200Immobilized on ND-L0.017 ± 0.0030.22 ± 0.04Mt-PPaseSoluble0.10 ± 0.011.3 ± 0.1Immobilized on ND-NH20.04 ± 0.010.53 ± 0.05Immobilized on ND-L0.17 ± 0.022.2 ± 0.3Conclusion:In this work, we applied the31P NMR spectroscopy to estimate the quantity of Piand PPiin synovial fluids of patients with CPPD disease. The conjugates of bacterial PPases with detonation nanodiamond were demonstrated to retain enzymatic activity in the hydrolysis of exogenous PPiin human synovial fluid. These results provide the basis for the further tests of PPase-based conjugates on animal modelsin vivo.References:[1]Rodina E.V., Valueva A.V., Yakovlev R.Y., Vorobyeva N.N., Kulakova I.I., Lisichkin G.V., Leonidov N.B. Immobilization of inorganic pyrophosphatase on nanodiamond particles retaining its high enzymatic activity // Biointerphases, Vol. 10, No. 4, 2015. P. 041005.Disclosure of Interests:Anastasiya Valueva: None declared, Roman Romanov: None declared, Sofia Mariasina: None declared, Maxim Elisеev Speakers bureau: Novartis, Menarini Group, Alium, Elena Rodina: None declared