RU2542411C1 - Conjugate of nanodiamond with pyrophosphatase and method of obtaining thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: conjugate represents nanodiamond particles with size 2-10 nm with pyrophosphorase, immobilised on them by means of linker, containing amino or amide groups. Content of pyrophosphatase constitutes 0.1-1 mg per 1 mg of nanodiamond, with specific activity of pyrophosphatase constituting to 95±5% of native pyrophosphatase activity. method of conjugate obtaining includes dissolution of nanodiamond with grafted hexamethylenediamine and/or nanodiamond aminated with ammonia in water, successive addition of water buffer solution HEPES with pH 7-8, magnesium chloride, sodium fluoride, sodium pyrophosphate, pyriphosphatase, and glutaraldehyde. After that, obtained mixture is exposed for 0.5-12 h, centrifuged, washed with water buffer solution Tris-HCl and dried.

EFFECT: obtaining conjugate of nanodiamond with pyrophosphatase, possessing increased stability.

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Description

The invention relates to the field of pharmacy and medicine, in particular, to pharmaceutical nanotechnology of enzymes, and relates to a conjugate of nanodiamond with pyrophosphatase for delivery of the enzyme to the body, as well as to a method for producing this conjugate and its variant.

The problem of treating pyrophosphate arthropathy (the common name for pseudogout) for many years has been extremely relevant in medicine. The disease belongs to the group of microcrystalline arthritis, characterized by multiple calcification of articular and periarticular tissues, mainly articular cartilage (chondrocalcinosis) due to the deposition of microcrystals of calcium pyrophosphate in them. The disease often develops in people over 55 years of age and is equally found in both men and women; 5% of the Earth’s population is exposed to it, and up to 27% at the age of 70 years and is accompanied by acute painful attacks [1]. Characteristic acute inflammation is due to the response of neutrophils that respond to calcium pyrophosphate crystals [2].

One of the most significant causes of pyrophosphate arthropathy is a violation of the production of enzymes responsible for the synthesis and / or transport of P ₂ O ₇ ^2- pyrophosphate anions into the intercellular matrix, for example, overproduction of the ANK transport protein or an increase in the activity of pyrophosphate-producing NPP enzymes (nucleotidyl pyrophosphorylases) [ 3].

There are no specific treatments for pseudogout to date. Treatment of concomitant diseases such as hemochromatosis, hyperparathyroidism and hypothyroidism does not lead to resorption of calcium pyrophosphate crystals, in rare cases, a decrease in the number of seizures is noted. In medical practice, for the treatment of pyrophosphate arthropathy, they try to use drugs for the treatment of gout, which with pyrophosphate arthropathy only relieve pain [4].

It is known to use additives of magnesium ions, including magnesium carbonate, as a prophylactic to reduce the frequency of acute attacks of pyrophosphate arthropathy [5-6]. In this case, changes in biochemical parameters and changes in the composition of the synovial fluid were not recorded and only a slight improvement in the condition of patients was shown. Research results are not used in practice.

The use of anti-inflammatory drugs, including non-steroidal anti-inflammatory drugs, corticosteroids and colchicine, is known in the clinic for the treatment of acute pseudogout and episodic inflammatory arthritis. Nonsteroidal anti-inflammatory drugs are essential in treating the disease, reducing the frequency of seizures. At the same time, they are unable to change the dynamics of the course of the disease, are not effective enough and are contraindicated for certain groups of patients [7]. Corticosteroids significantly alleviate the symptoms when using intra-articular injections, however, after applying moderate or high doses, they also show serious adverse reactions, such as hypertension, hyperglycemia, fluid retention and neuropsychic changes [7]. Colchicine is useful in relapsing pseudogout and in low doses to prevent crises [8], but it is often ineffective and causes many side effects: diarrhea, bone marrow depression, neuropathy, myopathy, skin rash similar to toxic epidermal necrolysis, and reversible azoospermia [ 7].

The ability of a probenecid drug to inhibit the growth of calcium pyrophosphate crystals in vitro is known [9]. However, it causes serious side effects: anorexia, urticaria, liver necrosis, nephrotic syndrome, leukopenia, etc. A study of the effect of probenecid on calcium pyrophosphate crystals in the human body has not been conducted [10].

It is known to use phosphocytrate to inhibit the growth of crystals of calcium pyrophosphate in vivo [11-12]. Phosphocytrate is an intracellzolar regulator of calcification and is capable of blocking pathological calcification [13]. Moreover, its functioning on in vivo models has not been studied, and there are no data on the toxicity of phosphocyrate and the safety of its use [10].

It is known to use methotrexate and anakinra drugs that affect the inflammasome to suppress immunity and relieve pain. Methotrexate can reduce the frequency and intensity of recurring bouts of pseudogout [14], as well as be an alternative drug in the prevention of hemochromatosis-associated arthropathy. However, at the same time, patients have an increased risk of side effects leading to liver diseases [8]. The action of anakinra is associated with the antagonism of the interleukin 1 receptor, which has a pro-inflammatory effect with pseudogout [15]. However, the drug fights only with the symptoms of the disease. In addition, anakinra has an immunosuppressive effect and high cost, which complicates its use in medical practice [8].

The above drugs and substances can be divided into two groups. The first group - do not affect the growth of crystals of calcium pyrophosphate and relieve only pain symptoms. The second group - inhibiting the growth of crystals of calcium pyrophosphate, but not reducing the concentration of this substance. At the same time, the substances and preparations used have a number of serious side effects, which so far has not led to their introduction into medical practice.

Therefore, the search and development of new highly effective and low-toxic agents for the treatment of pyrophosphate arthropathy is an important and extremely relevant medical and socially significant task.

The introduction of exogenous pyrophosphatase, an intracellular enzyme that catalyzes the hydrolysis of pyrophosphate to phosphate, can become a new promising area of therapy for this disease. This could allow the poorly soluble calcium pyrophosphate to be converted to more soluble calcium phosphate and, thus, directly reduce the deposition of calcium pyrophosphate salts during pyrophosphate arthropathy.

Pyrophosphatase is a magnesium-dependent enzyme, which is a globular protein and consists of several identical subunits with an active center [16].

However, with the introduction of pyrophosphatase into the affected tissues, the enzyme is rapidly inactivated by proteases and then removed from the body. This is due to the antigenicity of exogenously administered enzymes, in particular pyrophosphatase and the inability to create a high local concentration of the drug without increasing its total concentration.

It is also known that carbon nanoparticles of detonation nanodiamonds have a functionalized surface suitable for covalent inoculation of enzymes, are non-toxic, and biocompatible with the body [17].

It is known to use nanodiamonds to immobilize a number of substances of a protein nature, including enzymes, for use in medicine and biology as a carrier in drug delivery systems, biosensors, for the adsorption detection of biologically active substances, luminescent markers for studying cellular interactions, etc. [eighteen].

Known covalent vaccination of equine cytochrome C on a nanodiamond coated with poly-L-lysine. It was concluded that nanodiamonds are an excellent material for maintaining the stability of substances of protein nature [19].

Known conjugate of nanodiamonds with transferrin for receptor-mediated targeting of cancer cells for biomedical use [20]. The work indicates that, penetrating into the cells, the conjugate of a nanodiamond with transferrin can have a more directed effect compared to native transferrin.

The binding of trypsin and asparaginamidase F with nanodiamonds is known to increase their thermal and chemical stability [21].

Immobilization of the pyrophosphatase enzyme on nanodiamonds is not described in the patent and scientific literature.

An object of the present invention is to provide a pyrophosphatase-based agent with enhanced stability.

In accordance with the invention, a conjugate of nanodiamond with pyrophosphatase is described, which is particles of nanodiamonds 2-10 nm in size with pyrophosphatase immobilized on them by a linker containing amide or amino and amide groups, with a pyrophosphatase content of 0.1-1 mg per 1 mg nanodiamond and specific pyrophosphatase activity up to 95 ± 5% of the activity of native pyrophosphatase.

As the linker, glutaraldehyde and / or hexamethylenediamine can be used. The linker is predominantly NH — C (O) - (CH ₂ ) ₂ —C (O) - or —NH (CH ₂ ) ₆ —NH — C (O) - (CH ₂ ) ₂ —C (O) -.

Also described is a method for producing a nanodiamond conjugate with pyrophosphatase, characterized in that the nanodiamond with a grafted hexamethylenediamine or ammonia-aminated nanodiamonds with a particle size of 2-10 nm are dissolved in water to form a suspension, and an aqueous buffer of HEPES hydroxyethyl piperazine-N-2-nitric acid is added sequentially. ) with a pH of 7-8, magnesium chloride, sodium fluoride, sodium pyrophosphate, pyrophosphatase and glutaraldehyde, followed by maintaining the mixture for 0.5-12 hours, centrifugation, washing with water buffer Tris-HCl to pH 7-8 and drying.

The process is carried out at a weight ratio of pyrophosphatase: nanodiamonds with grafted hexamethylenediamine or aminated nanodiamonds: glutaraldehyde 1: 1-10: 5-50, respectively.

A method of obtaining the described conjugate consists in immobilizing pyrophosphatase on a nanodiamond by covalently linking pyrophosphatase to the primary amino group of the linker grafted onto the surface of the nanodiamond.

In more detail, the method is as follows. To obtain a nanodiamond containing primary amino groups, it is annealed in a stream of hydrogen gas at a temperature of 500-1200 ° C for 1-8 hours, then subjected to liquid-phase chlorination with molecular chlorine under photochemical exposure to visible light at a temperature of 50-70 ° C for 36- 60 hours, followed by washing with carbon tetrachloride, centrifugation and drying under vacuum. Chlorine-modified nanodiamonds are suspended in absolute alcohol, hexamethylenediamine is added, and in the presence of the Hunig reagent, they are boiled for 24 hours, followed by washing with absolute alcohol and drying under vacuum. The obtained nanodiamond with grafted hexamethylenediamine (containing primary amino groups) is dissolved in water to form a suspension and HEPES buffer solution with a pH of 7-8, magnesium chloride to a concentration of 2 mM, sodium fluoride to a concentration of 10 mM, sodium pyrophosphate to a concentration of 100 μM, pyrophosphatase are successively added. to a concentration of 0.1-1 mg / ml and glutaraldehyde to a concentration of 50-500 μM and the resulting mixture is kept for 30 minutes to 12 hours, followed by centrifugation, washing with Tris-HCl buffer solution at pH 7-8, and centrifugation again iem slurry by decanting the supernatant solution and drying.

A variant of the described method for producing immobilized pyrophosphatase is distinguished by the preparation of a nanodiamond containing primary amino groups and the absence of an additional linker between the nanodiamond and the enzyme. For this, a chlorine-modified nanodiamond obtained by the method described above is subjected to amination in a stream of ammonia gas at a temperature of 400-500 ° C for 2-4 hours. The obtained aminated nanodiamond (containing primary amino groups) is chemically coupled directly with pyrophosphatase in the same way as described above.

Figure 1 shows the IR spectrum of the conjugate immobilized on a nanodiamond pyrophosphatase in the range of 1800-400 cm ^-1 . The spectrum contains two bands of medium intensity with maximums at 1660-1630 and 1560-1510 cm ^-1 and weak signals with maximums at 1458, 1184 cm ^-1 , as well as two wide bands from 1320 to 900 cm ^-1 and from 870 to 400 cm ^-1 .

IR spectra were recorded on a FTIRS IR200 Thermonicolet instrument with a resolution of 2 cm ^-1 , the number of scans was 100. For analysis, weighed samples were mixed with KBr powder and pressed into a tablet.

An estimate of the amount of the enzyme during its immobilization on the nanodiamond was determined by the difference in the concentration of the enzyme in the solution before and after immobilization.

The pyrophosphatase concentration in the solution was determined on an Ultrospec 3000 spectrophotometer (Pharmacia Biotech, Sweden), measuring the absorbance at a wavelength of 280 nm and comparing the intensity with the intensity of the pyrophosphatase solution with a known concentration.

The enzymatic activity of pyrophosphatase was evaluated by the rate of formation of phosphate from pyrophosphate by continuously measuring its concentration on a “Semi-automatic phosphate analyzer” [22]. The determination is based on a shift in the absorption maximum of the methyl green solution (632 nm) to the longer wavelength region (656 nm) that occurs when the molybdate ion is bound.

The stability of the resulting conjugate was evaluated by the change in its activity by aging at 75 ° C for 4.5 hours

Figure 2 shows the thermal inactivation curves of the conjugate of pyrophosphatase immobilized on a nanodiamond, having a two-phase dependence. For native pyrophosphatase, the nature of the curve is explained by the oligomeric nature of the protein: the first phase of inactivation corresponds to dissociation of the oligomeric protein, and the second to denaturation. The abscissa shows the logarithmic time scale of the incubation of the compositions, min. On the ordinate axis - their specific activity at a time,% of the original. Comparison of the denaturation rate constants (Table 1) shows that the rate of denaturation of native pyrophosphatase is higher, therefore, the stability of samples immobilized on nanodiamond pyrophosphatase is higher and exceeds the native enzyme by 1.5 times.

Table 1. Thermal inactivation rate constants of native and immobilized pyrophosphatase on (ND) nanodiamonds. Options Sample Pyrophosphatase HA-NH (CH ₂ ) ₆ NH-E-pyrophosphatase HA-NH-pyrophosphatase Speed constant, min ^-1 I phase 0.31 ± 0.05 0.15 ± 0.01 0.15 ± 0.01 II phase (56 ± 5) · 10 ^-4 (39 ± 4) · 10 ^-4 (38 ± 4) · 10 ^-4 Note *: E = —C (O) - (CH ₂ ) ₂ —C (O) -.

The obtained conjugate of pyrophosphatase immobilized on a nanodiamond has increased stability and can be used in medicine as a system for delivering an enzyme to the body.

A brief description of the graphic materials.

Figure 1. The IR spectrum of the described conjugate immobilized on a nanodiamond pyrophosphatase in the region of 1800-400 cm ^-1 .

Figure 2. Thermal inactivation curves of the described pyrophosphatase immobilized on a nanodiamond compared to native pyrophosphatase. 1 - native pyrophosphatase; 2 - nanodiamond-NH-C (O) - (CH2) 2-C (O) pyrophosphatase; 3 - nanodiamond-NH (CH2) 6-NH-C (O) - (CH2) 2-C (O) pyrophosphatase.

The invention is illustrated by the following examples.

Example 1

A weighed portion of the initial nanodiamond weighing 500 mg is annealed in a stream of hydrogen gas at a rate of 3.0 l / h at a temperature of 800 ° C for 6 h. Then, the annealed nanodiamond is subjected to liquid phase chlorination with molecular chlorine dissolved in 250 ml of CCl ₄ to 5.6% by weight . Cl ₂ . The chlorination reaction is carried out by photochemical exposure to visible light for 48 hours at a temperature of 60 ° C. The sample was then washed with CCl ₄ by centrifuging the suspension at 6000 rpm and dried under a pressure of 0.1 mm. Hg to constant weight. A sample of chlorinated nanodiamond weighing 400 mg is dissolved in 50 ml of absolute alcohol, a sample of hexamethylenediamine (800 mg) and 5 drops of Hunig's reagent (N, N-diisopropylethylamine) are added. The mixture is boiled for 24 hours at 80 ° C and then repeatedly washed with water and absolute alcohol and dried under pressure to constant weight. A portion of the obtained 25 mg hexamethylenediamine grafted nanodiamond is dissolved in 25 ml of water to form a suspension and HEPES buffer solution with a pH of 7-8 is added sequentially, magnesium chloride to a concentration of 2 mM, sodium fluoride to a concentration of 10 mM, sodium pyrophosphate to a concentration of 100 μM, pyrophosphatase isolated from E. Coli to a concentration of 1 mg / ml and glutaraldehyde to a concentration of 500 μM and the resulting mixture was kept at room temperature with constant stirring for 6 hours, followed by centrifugation, washing with buffer with a 50 mM Tris-HCl solution containing 2 mM MgCl ₂ , re-centrifuging the suspensions at 14,000 rpm, decanting the supernatant and drying.

The resulting product is a gray powder with a moisture content of 4.5%, characterized by a PC spectrum in the range of 1800-400 cm ^-1 : two bands of medium intensity with maxima at 1660-1630 and 1560-1510 cm ^-1 and weak signals with maxima at 1458, 1184 cm ^-1 , two wide stripes from 1320 to 900 cm ^-1 and from 870 to 400 cm ^-1 (Figure 1).

The amount of pyrophosphatase immobilized on a nanodiamond is determined by the difference in the concentration of the enzyme in the solution before and after immobilization. The concentration of pyrophosphatase immobilized on a nanodiamond is determined spectrophotometrically by measuring absorbance at a wavelength of 280 nm. A standard solution of pyrophosphatase with a concentration of 1 mg / ml with an optical path length of 1 cm has an absorption of 1.18 OU. The solution after the process of immobilization with an optical path length of 1 cm has an absorption of 1.18 OU. Therefore, the amount of pyrophosphatase immobilized on a nanodiamond is 1 mg of pyrophosphatase per 1 mg of nanodiamond.

The preparation of pyrophosphatase immobilized on a nanodiamond according to the method described above at a concentration of glutaraldehyde of 50 μM for 30 minutes leads to the content of immobilized pyrophosphatase 0.1 mg per 1 mg of nanodiamond.

The determination of pyrophosphatase activity is based on a shift in the absorption maximum of the methyl green solution (632 nm) to the longer wavelength region (656 nm), which occurs upon binding of the phosphomolybdate ion resulting from the interaction of molybdate and phosphate synthesized from pyrophosphate.

Pyrophosphatase activity is determined at a temperature of 20 ° C by the rate of formation of phosphate from pyrophosphate by continuously measuring the concentration of phosphate on a "Semi-automatic phosphate analyzer" [22] and is calculated by the formula

,

,

where a is the activity of pyrophosphatase in IU / mg;

V _cell is the volume of the reaction mixture (ml);

V _E is the volume of an aliquot of the enzyme added to the cell (μl);

[E] is the concentration of the enzyme (mg / ml);

k is the conversion coefficient determined in a separate experiment, equal to the phosphate concentration (in μM), which gives the response of the recorder to 1 mm;

tgα is the measured initial rate of phosphate ion accumulation (mm / min).

The specific activity of the enzyme was expressed in international units (IU / mg). 1 IU / mg corresponds to the conversion rate of 1 micromole of the substrate in 1 minute under the action of 1 mg of the enzyme.

The activity of the native enzyme calculated by formula (1) was 222 IU / mg.

To determine the activity of pyrophosphatase immobilized on a nanodiamond, before feeding the phosphate analyzer to the analyzer, the analyte containing pyrophosphatase immobilized on a nanodiamond was diluted to an enzyme concentration of 2.7 μg / ml and a 5 μl aliquot was added to the 10 ml reaction mixture with constant stirring on a magnetic stirrer. The reaction mixture contained a buffer of 50 mm Tris-HCl, 100 μm magnesium pyrophosphate and 5 mm MgCl ₂ . After introducing aliquots of the enzyme into it, the reaction mixture was fed into a “Semi-automatic phosphate analyzer”, where it was mixed with a solution of ammonium molybdate and sulfuric acid ((NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O - 24 g, H ₂ SO _{4 conc.} - 77 ml, water - 2 l), and then with a solution of methyl green with Triton X-305 (methyl green - 150 mg, Triton X-305 - 6.8 g, water - up to 2

To determine the activity of pyrophosphatase immobilized on a nanodiamond, before feeding the phosphate analyzer to the analyzer, the analyte containing pyrophosphatase immobilized on a nanodiamond was diluted to an enzyme concentration of 2.7 μg / ml and a 5 μl aliquot was added to the 10 ml reaction mixture with constant stirring on a magnetic stirrer. The reaction mixture contained a buffer of 50 mm Tris-HCl, 100 μm magnesium pyrophosphate and 5 mm MgCl ₂ . After introducing aliquots of the enzyme into it, the reaction mixture was fed into a “Semi-automatic phosphate analyzer”, where it was mixed with a solution of ammonium molybdate and sulfuric acid ((NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O - 24 g, H ₂ SO _{4 conc.} - 77 ml, water - 2 l), and then with a solution of methyl green with Triton X-305 (methyl green - 150 mg, Triton X-305 - 6.8 g, water - up to 2 l). The optical density of the resulting solution of the phosphomolybdate complex was measured in a flow cell and recorded on a recorder. The hydrolysis rate was determined by the slope of the straight line, reflecting the accumulation of phosphate in the cell over time (tgα). The phosphate concentration was determined by a pre-constructed calibration curve, which for 89 μM phosphate solution was 89 mm of the line length of the recorder. The activity of pyrophosphatase immobilized on a nanodiamond was calculated by the formula (1)

The activity of pyrophosphatase immobilized on a nanodiamond was calculated by the formula

The activity of pyrophosphatase immobilized on a nanodiamond was 212 IU / mg or 95 ± 5% of the activity of native (unbound) pyrophosphatase.

To study the stability of pyrophosphatase immobilized on a nanodiamond compared to native pyrophosphatase, 1 ml of a buffer solution containing 50 mM Tris-HCl and pyrophosphatase immobilized on a nanodiamond with a concentration of 8 μg / ml was kept in a thermostat at a temperature of 75 ° C for 4.5 h and after at certain time intervals (from 10 min to 1 h), an aliquot of the solution was taken and the activity of pyrophosphatase immobilized on nanodiamond was measured by the method described above. In the control experiment used 1 ml of a buffer solution of 50 mm Tris-HCl and native pyrophosphatase in the same concentration (8 μg / ml). To determine the kinetic parameters of temperature inactivation, nonlinear regression was performed using the SigmaPlot program according to the equation

Where a and c are the drop in catalytic activity in phases I and II, respectively;

b and d are the rate constants of phases I and II, respectively.

The stability of pyrophosphates immobilized on a nanodiamond was evaluated by the denaturation rate constant equal to (39 ± 4) · 10 ⁴ , which is 1.5 times lower than the native pyrophosphatase denaturation rate constant (56 ± 5) · 10 ^-4 . Thus, the stability of pyrophosphatase immobilized on a nanodiamond is 1.5 times higher than the stability of native pyrophosphatase.

Example 2

A weighed portion of the initial nanodiamond weighing 500 mg is annealed in a stream of hydrogen gas at a rate of 3.0 l / h at a temperature of 800 ° C for 6 h. Then, the annealed nanodiamond is subjected to liquid phase chlorination with molecular chlorine dissolved in 250 ml of CCl ₄ to 5.6% by weight . Cl ₂ . The chlorination reaction is carried out by photochemical exposure to visible light for 48 hours at a temperature of 60 ° C. The sample was then washed with CCl ₄ by centrifuging the suspension at 6000 rpm and dried under a pressure of 0.1 mm. Hg to constant weight. A sample of chlorinated nanodiamond weighing 250 mg is annealed in a stream of ammonia gas at a rate of 3.0 l / h at a temperature of 350 ° C for 4 hours. A portion of the obtained aminated nanodiamond weighing 25 mg is dissolved in 25 ml of water to form a suspension and HEPES buffer solution is added sequentially. with a pH of 7-8, magnesium chloride to a concentration of 2 mM, sodium fluoride to a concentration of 10 mM, sodium pyrophosphate to a concentration of 100 μM, pyrophosphatase isolated from E. Coli to a concentration of 1 mg / ml and glutaraldehyde to a concentration of 50 μM and incubated the resulting mixture at room temperature with constant stirring for 6 h followed by centrifugation, washing with buffer solution of 50 mM Tris-HCl, containing 2 mM MgCl _2, re-suspensions by centrifugation at 14,000 rev / min, the supernatant solution by decantation and drying.

The resulting product was a gray powder with a moisture content of 4.7%, characterized by an IR spectrum in the range of 1800-400 cm- ¹ : two medium-intensity bands with peaks at 1660-1630 and 1560-1510 cm ^-1 and weak signals with peaks at 1458, 1184 cm ^-1 , two wide stripes from 1320 to 900 cm ^-1 and from 870 to 400 cm ^-1 (Figure 1).

The amount of pyrophosphatase immobilized on a nanodiamond is determined by the difference in the concentration of the enzyme in the solution before and after immobilization, as in Example 1. A standard solution of pyrophosphatase with a concentration of 1 mg / ml with an optical path length of 1 cm has an absorption of 1.18 OU. The solution after the process of immobilization with an optical path length of 1 cm has an absorption of 0.73 OE. Therefore, the amount of pyrophosphatase immobilized on a nanodiamond is 0.62 mg of pyrophosphatase per 1 mg of nanodiamond.

The determination of the activity of pyrophosphatase was carried out according to the method of example 1. The activity of the native enzyme calculated by the formula (1) was 222 IU / mg.

To determine the activity of pyrophosphatase immobilized on a nanodiamond, before feeding the phosphate analyzer to the analyzer, the analyte containing pyrophosphatase immobilized on a nanodiamond was diluted to an enzyme concentration of 6.8 μg / ml and an aliquot of 10 μl was added to the 10 ml reaction mixture with constant stirring on a magnetic stirrer.

The activity of pyrophosphatase immobilized on a nanodiamond was calculated by the formula

The activity of pyrophosphatase immobilized on a nanodiamond was 141 IU / mg or 62 ± 5% of the activity of native (unbound) pyrophosphatase.

A study of the stability of pyrophosphatase immobilized on a nanodiamond was carried out according to the procedure of Example 1.

The stability of pyrophosphates immobilized on a nanodiamond was estimated by the denaturation rate constant equal to (38 ± 4) · 10 ⁴ , which is 1.5 times lower than the rate constant of denaturation of native pyrophosphatase (56 ± 5) · 10 ^-4 . Thus, the stability of pyrophosphatase immobilized on a nanodiamond is 1.5 times higher than the stability of native pyrophosphatase.

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Claims (3)

1. The conjugate of nanodiamond with pyrophosphatase for the delivery of pyrophosphatase into the body, which is particles of nanodiamonds 2-10 nm in size with a linker containing amide or amino and amide groups immobilized with pyrophosphatase, with a pyrophosphatase content of 0.1-1 mg per 1 mg of nanodiamond and specific pyrophosphatase activity up to 95 ± 5% of the activity of native pyrophosphatase. 2. The conjugate according to claim 1, where the linker is —NH — C (O) - (CH ₂ ) ₂ —C (O) - or —NH (CH ₂ ) ₆ —NH-C (O) - (CH ₂ ) ₂ -C (O) -. 3. The method for producing the conjugate according to claim 1, characterized in that the nanodiamond with grafted hexamethylenediamine or ammonia-aminated nanodiamonds with a particle size of 2-10 nm are dissolved in water to form a suspension and an aqueous HEPES buffer solution with a pH of 7-8 is added sequentially, magnesium chloride , sodium fluoride, sodium pyrophosphate, pyrophosphatase and glutaraldehyde at a weight ratio of pyrophosphatase: nanodiamonds with grafted hexamethylenediamine or aminated nanodiamonds: glutaraldehyde 1: 1-10: 5-50, respectively, followed by aging the resulting mixture for 0.5-12 hours, centrifugation, washing with an aqueous buffer solution of Tris-HCl with a pH of 7-8 and drying.

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