Method of determining biological inequivalence of nanodiamonds

FIELD: chemistry.

SUBSTANCE: incubation medium, oxidation substrate and tetraphenylphpsphonium chloride as indicator are placed into respective measuring cell of installation for measuring mitochondria potential, provided with tetraphenylphosphonium-selective electrode, change of tetraphenylphosphonium concentration is registered and when constant tetraphenylphosphonium concentration is achieved, mitochondria, isolated from animal organism, are added. Change of membrane potential of mitochondria is registered by change of electrode signal, when constant potential is achieved, respective water suspensions of analysed samples of nanodiamonds with pH 7.2-7.4 are added, and value of rate of change of mitochondria membrane potential is measured. Presence of statistically reliable difference of rates of mitochondria membrane potential change testifies to biological inequivalence of compared samples of nanodiamonds.

EFFECT: expressive and available method of determining biological inequivalence of nanodiamonds.

2 cl, 1 tbl, 1 dwg, 1 ex

 

The present invention relates to the field of pharmacology, biopharmaceutics and pharmaceutical industry and relates to a method of determining the biological neweventnotify samples of nanodiamonds, which can find application in the quality control of industrially produced and modified nanodiamonds, as well as in the production of pharmaceuticals and drug delivery based on the nd.

Nanodiamonds are a distinct group of carbon nanoparticles. A special place among them is occupied by detonation nanodiamonds, which are obtained by detonation of explosives or their mixtures in a special chamber. As a result of detonation is formed mixture, the composition of which comprises particles of nd. To highlight nd the mixture is subjected to oxidation treatment in harsh environments [1].

Received detonation nanodiamond has a number of distinctive physical and chemical characteristics: ultra-small size of the primary particles (4-6 nm), highly developed specific surface (up to 400 m2/g), high concentration of functional groups therein, and chemically inert core particles [1].

There are several technology options for isolation and purification of detonation nanodiamond, each of which affects the physico-chemical characteristics of the resulting particles and can affect their biological properties. Cu is IU, it is known that further chemical modification of nanodiamond also affect its biological properties.

So, in [2] studied the toxicity of samples of nanodiamonds brand "ultraFine Diamond" (Russia) and manufactured by General Electric (USA) on-line lung epithelium adenocarcinoma human A. Nano-diamond mark "ultraFine Diamond" (Russia) obtained by the method of detonation synthesis, and the final stage of cleaning can vary from treatment with perchloric acid, potassium permanganate prior to cleaning with nitric acid [3]. Therefore, the final properties of the brand of nd brand "ultraFine Diamond", depending on the stage of its final treatment, can be different [3]. Nanodiamond manufactured by General Electric (USA) obtained by the method of static synthesis with subsequent acid treatment [4]. If nd made in the USA of cell survival was higher than that of nd, produced in Russia. In turn, the modification (oxidation of the mixture of acids) samples nd these two brands leads to a decrease of cell survival [3].

In [5] was used nanodiamonds obtained in the NanoCarbon Research Institute Co., Ltd (Japan). Technology data of nanodiamonds includes oxidation process, and then grind nanodiamond powder in a ball mill using Zirconia balls [6]. It is shown that the survival of the notches in the case of processing the nanodiamond is 80-90% compared with control. Subsequent surface modification of nanodiamond polyethylene glycol reduces the survival rate of cells to 70-80% and its amination of up to 55% [5].

Mandatory application of nanodiamond in medicine as carrier systems for drug delivery is the identity of its chemical and physico-chemical and pharmacotoxicological characteristics. For the nd in biomedical applications of the above works [2-6] clearly implies the need to find and develop ways of controlling non-bioequivalent nanodiamonds obtained by different technologies and undergo different chemical treatment, including chemical modification.

Currently in the patent and scientific literature does not describe how to identify differences in biological effects and biological neweventnotify samples of carbon nanoparticles, including nanodiamonds obtained using various techniques and/or different ways of chemically modified.

The aim of the invention is to develop a rapid and affordable method for determining the biological neweventnotify samples nd.

The inventive method consists in the comparative determination of the effect of samples nd on the membrane potential of mitochondria of rat liver. The essence of what the procedure is the following. In the measuring cell make a mixture of freshly isolated rat liver mitochondria, substrate oxidation and the indicator are added, and the suspension of nd and record the rate of change of the concentration of free indicator, which determines the change in membrane potential of mitochondria. On the basis of comparison of velocity changes of membrane potentials, obtained by the action of the analyzed and reference materials, to judge the presence or absence of biological neweventnotify these samples. As a standard sample (reference sample) can be selected by any of the studied sample nd depending on the purpose of its further use.

Mitochondria are the Central link in the chain of energy for cellular processes, which in the presence of substrate oxidation (e.g., succinate sodium/potassium pyruvate sodium/potassium, and others) actively generate the membrane potential inside the membrane [7]. To determine the membrane potential use indicators that respond to changes in its size. As these indicators can serve a variety of hydrophobic ions (tetraphenylphosphonium, tetraphenylboron) and various probes that are sensitive to surface and transmembrane potential (for example, rhodamine-123, oxonol-6, N,N-dimethyl-N-nonyl-N-temperaturebased and others) [8]. Known is about, that link membrane potential of mitochondria (Δφ) with the concentration of free (recordable) form of the indicator (Coutand concentration of the indicator inside the mitochondria (Cin) is described by the Nernst equation [9]:

Δφ=(RT/zF)×ln(Cin/Cout),

where R is the universal gas constant, equal 8,31 j/(mol·K);

T is the absolute temperature (K);

z is the charge of an ion (for tetraphenylporphine z=1);

F - Faraday constant, equal 96485,35 KL/mol.

From the Nernst equation, it follows that with increasing membrane potential indicator, for example tetraphenylphosphonium, begins to penetrate into the mitochondria, the concentration of the free form in the cell is reduced. With decreasing membrane potential, on the contrary, there is a release indicator from the internal space of the mitochondria, thereby increasing the amount of free indicator.

To assess the impact of nd on the membrane potential of mitochondria we propose to use the value not the membrane potential, and the speed of its change, which is measured by changing the value of the concentration of free indicator in the cell per unit of time (min.)

The inventive method are described in more detail in the following. To compare samples of nd, taken in an amount of 0.1-0.25 mg add 2 ml of distilled water. Adding the Astor potassium hydroxide bring the pH of the suspension to a value of 7.2 to 7.4. The measurements were carried out using potentiometric computerized installation tetraphenylphosphonium-selective electrode [10]. In the measuring cell is placed 20 μl of mitochondria isolated from liver homogenate of rats according to standard methods [11], which is that of mitochondria isolated from the liver of adult male Wistar rats in accordance with standard procedure, including differential centrifugation and storage of selected mitochondrial ice [11].

Incubation of mitochondria in all samples is carried out in a standard environment: 125 mm potassium chloride, 15 mm 4-(2-oxyethyl)1-piperazineethanesulfonic acid, 1,5 mm phosphate at pH of 7.25 in the presence of 4 mm potassium succinate. Add indicator (up to a concentration of 1 μm). Then add an aliquot (10-50 µl) of the suspension sample nd and record the rate of change of the concentration of free indicator. According to the measurement results build graphs of the effect of the compared samples of nd on the membrane potential in the coordinate concentration of free indicator - time” (Fig.1).

To add to the cell mitochondria suspensions of the samples of the inherent nature of all curves practically do not differ. It is significant curves after adding to the mitochondria of samples nd (in Esenia which is marked in Fig.1 by arrows). Choose the linear portion of the curve after addition of the sample and calculate the change in the concentration tetraphenylporphine in time, expressing the result as the rate of change in concentration of the indicator in a minute.

On the basis of statistically significant comparisons (±0,025 - the measurement of these quantities make a conclusion about the presence or absence of biological neweventnotify compared samples of nd.

A brief description of graphic materials.

Fig.1. Decline curves membrane potential of mitochondria under the influence of industrial samples and modified samples nd (time additive samples of nd to the mitochondria marked by arrows). 1 - control; 2 - hydrogenated nanodiamond brand "UDA-TAN; 3 - nano-diamond mark "UDA-TAN; 4 - nano-diamond mark "Standard ND"; 5 - chlorinated nano-diamond mark "UDA-TAN".

The invention is illustrated by the following example.

Example.

According to the claimed method was analyzed 4 sample nanodiamonds, including two samples of industrial nd: nd brand "UDA-TAN" (SKTB "Technologist", Russia) and nano-diamond mark "ND Standart" ("Adamas Nanotechnologies, USA) (samples 3 and 4, respectively). Nano-diamond mark “UDA-TAN obtained by detonation synthesis with stage cleaning 50-60% nitric acid at a temperature of 230-240°C and a pressure of 80-90 ATM and followed what ammonolysis under pressure at 200°C (pH=10) [1, page 76-108].

Details of the production technology nd brand "ND Standart" is unknown.

Nanodiamonds with gidrirovannoe (sample 2) and chlorinated (sample 5) surfaces were obtained by chemical modification of the sample 3 in accordance with the methods of [12].

Mitochondria isolated from the liver of adult male Wistar rats in accordance with the standard procedure by differential centrifugation [11]. All requirements for the care and working with animals were carefully observed. Liver was homogenized in ice buffer containing 70 mm sucrose, 10 mm 2-amino-2-hydroxymethyl-propane-1,3-diol and 1 mm ethylene glycol-bis(2-aminoethylamide ether)-N,N,N,N-tetraoxane acid (pH of 7.4). The homogenate was centrifuged at 600 g for 7 minutes at 4°C, were selected fraction of the supernatant and centrifuged at 9000 g for 10 min to sediment the mitochondria. Mitochondria were twice washed in the above medium without ethylene glycol-bis(2-aminoethylamide ether)-N,N,N,N-tetraoxane acid and centrifuged. Next, the precipitate of mitochondria containing 60 mg of protein, suspended in wash medium and kept on ice.

As a control, we measured the membrane potential of mitochondria without adding nanodiamonds (curve 1 in Fig.1). To compare samples of nd, each of which took in an amount of 0.1 mg, EXT is ulali 2 ml of distilled water, thoroughly mixed, the mixture and adding a solution of potassium hydroxide brought the pH of the resulting suspension to a value of 7.4.

For analysis of each sample in the cell was placed 20 ml of mitochondria, isolated by the method of [10]. An indicator used chloride solution tetraphenylporphine, which added to the value of its concentration in the mixture equal to 1 μm. Further, as the substrate oxidation used succinate potassium and record the concentration of free tetraphenylporphine in the measuring cell. Then for each sample inherent in the measuring cell was added 30 μl of the previously prepared suspension and recorded the change in the concentration tetraphenylporphine on the linear parts of the curves after addition of the samples of nd (Fig.1). Next chose the linear region of the curve after addition of the sample, and calculated the change in the concentration tetraphenylporphine in time, expressing the result as the rate of change in concentration of the indicator in a minute. The measurement was repeated in strictly identical conditions not less than three times. Based on the obtained values of the rate of change of concentration tetraphenylporphine for each sample to calculate the average value and the magnitude of the statistical error. On the basis of statistically significant differences in the velocities is modify the concentration of the indicator for different samples nd did the conclusion of their biological neweventnotify. The results of the measurements are shown in the Table.

As the comparison sample used sample nd Russian production (sample 3).

Table
The results of the experiment to determine bioequivalence samples nd () (relative to the sample 3) of the claimed method.
No. sampleSampleThe rate of change of concentration of the indicator, µm/minBioequivalents
1Control0,021±0,01-
2Hydrogenated nanodiamond brand "UDA-TANG"0,025±0,025No
3Nano-diamond mark "UDA-TAN" (reference sample)0,035±0,025-
4Nano-diamond mark "Standard ND"0,377±0,025
5 Chlorinated nano-diamond mark "UDA-TANG"0,284±0,025

From the Table it follows that the speed of decrease of the membrane potential in the event of exposure of samples of industrial nd Russian production and nd with gidrirovannoe surface is 0,035±0.025 and 0,025±0,025 µm/min, respectively. As the speed difference not statistically significant, it was concluded on biological equivalence of samples 2 and 3 (i.e., about the lack of bioequivalence). At the same time, the speed of decrease of the membrane potential for samples 4 and 5 is 0,377±0.025 and 0,284±0,025 µm/min, respectively, which, given the statistically significant differences in the velocities, allows to make a conclusion about their biological neweventnotify not only among themselves but also with respect to other samples of nanodiamonds.

References

1. C. Y. Dolmatov. Detonation nanodiamonds. Production, properties, application. / St. Petersburg: Izd. NGO "Professional", 2011. 536 S.

2. K.-K. Liu, C.-L. Cheng, C.-C. Chang and Ju.-I Chao. Biocompatible and detectable carboxylated nanodiamond on human cell // Nanotechnology. 2007. V. 18. 325102 (10 pp.).

3. F. Huang, Yi Tong, Sh. Yun. Synthesis Mechanism and Technology of Ultrafine Diamond from Detonation // Physics of the solid body. 2004. So 46. No. 4. S. 601-604.

4. C. B. Muratov, A. A. Vasiliev, V. C. Matias, I. I. Duda. The influence of gas-forming impurities on the heat capacity of Nanoka the metallic diamond detonation synthesis // Nanostructured materials. 2011. No. 1. S. 23-31.

5. X.-Q. Zhang, M. Chen, R. Lam, X. Xu, E. Osawa, D. Ho. Polymer-Functionalized Nanodiamond Platforms as Vehicles for Gene Delivery // ACS Nano. 2009. V. 3. N. 9. P. 2609-2616.

6. E. Osawa. Remarks on the handling of colloidal solutions of 5-nm diamond particles / NCRI Technical Bulletin, 2009. No..3. P. 1-8.

7. D. Nelson, M. Cox. Fundamentals of biochemistry Lehninger: 3 so So 2: Bioenergetics and metabolism. / Lane. from English. - M.: BINOM. Knowledge laboratory. 2014. S. 306-342.

8. N. Hennis. Biomembranes. Molecular structure and function. / M.: Mir. 1997. S. 319-321.

9. N. Kamo, M. Muratsugu, R. Hongoh, and Y. Kobatake. Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state // J. Membrane Biol. 1979. V. 49. P. 105-121.

10. N. And. Paducheva In. A. Teplova, N. In. Beloborodov. Role of thiol antioxidants in the restoration of mitochondrial function, the modified microbial metabolites. // Biophysics. 2012. So 57. No. 5. S. 820-826.

11. N. I. Fedotcheva, R. E. Kazakova, M. N. Kondrashova, N. V. Beloborodova. Toxic effects of microbial phenolic acids on the functions of the mitochondria // Toxicology Letters. 2008. V. 180. P. 182-188.

12. G. V. Lisichkin, I. I. Kulakova, A. Yu. Gerasimov, A. V. Karpukhin, R. Yakovlev Yu. Halogenation of detonation-synthesis nanodiamond surface // Mendeleev Communication. 2009. No. 19. P. 309-310.

1. The method of determining the biological neweventnotify of nanodiamonds by comparative determine the effect of sample nd on the membrane potential of the mitochondria of animals, namely, that corresponding to the measuring cell unit for measuring the capacity of the mitochondria, is equipped with tetrafen lovoni-selective electrode, put the incubation medium containing an aqueous solution of potassium chloride, 4-(2-oxyethyl)1-piperazineethanesulfonic acid, phosphate buffer, substrate oxidation and as an indicator of chloride tetraphenylporphine, register the change of concentration tetraphenylporphine and when constant concentration tetraphenylporphine add isolated from animals mitochondria change signal electrode register the change of membrane potential of mitochondria, while achieving constant potential add appropriate water suspensions of the samples of nanodiamonds with a pH of 7.2-7.4 and measure the magnitude of the rate of change of membrane potential of mitochondria, the presence of statistically significant differences in the speed change of the membrane potential of mitochondria indicates biological neweventnotify compared samples of nanodiamonds.

2. The method according to p. 1, where the substrate oxidation using potassium succinate.



 

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EFFECT: invention ensures higher accuracy of measurements.

3 cl, 6 dwg

Pcd diamond // 2522028

FIELD: process engineering.

SUBSTANCE: invention relates to PCD diamond to be used in production of water-jet ejectors, engraving cutters for intaglio, scribers, diamond cutters and scribing rollers. PCD diamond is produced by conversion and sintering of carbon material of graphite-like laminar structure at superhigh pressure of up to 12-25 GPa and 1800-2600°C without addition of sintering additive of catalyst. Note here that sintered diamond grains that make this PCD diamond feature size over 50 nm and less than 2500 nm and purity of 99% or higher. Diamond features grain diameter D90 making (grain mean size plus grain mean size × 0.9) or less and hardness of 100 GPa or higher.

EFFECT: diamond features laminar or fine-layer structure, ruled out uneven wear, decreased abrasion.

15 cl, 5 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention may be used in medicine in producing preparations for a postoperative supporting therapy. What is involved is the high-temperature decomposition of methane on silicone or nickel substrate under pressure of 10-30 tor and a temperature of 1050-1150°C. The heating is conducted by passing the electric current through a carbon foil, cloth, felt or a structural graphite plate whereon the substrates are arranged. An analogous plate whereon a displacement potential from an external source is sent is placed above the specified plate. Nanodiamonds of 4 nm to 10 nm in size are deposited on the substrates.

EFFECT: higher effectiveness of the method.

1 dwg, 6 ex

FIELD: process engineering.

SUBSTANCE: invention relates to blast processes of the synthesis of materials, in particular, diamonds. Proposed device comprises flow vessel 1 with tight cover 3, mix of explosive arranged inside said vessel that features a high specific energy and graphite or carbon-bearing explosive with negative oxygen balance, initiator 5, indestructible cylindrical barrier 6 composed by pipe arranged aligned with vessel 1 there inside. Note here that said mix of graphite and explosive and initiator 5 are placed at barrier 6 centre.

EFFECT: protection of device wall against maximum loads, increased bulk of explosive without increase in device volume and weight.

1 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to pharmacology, nanomaterials and nanotechnology, and concerns a method for selective final purification of nanodiamonds to remove foreign nitrate ions and sulphur compounds to be used in pharmaceutics; the method implies that charge-free nanodiamond powder is treated with alkaline water of the concentration of 0.01-1 mole/l at 20-100°C; the prepared suspension is then decanted and centrifuged; the precipitation is washed with water using ultrasound, separated and dried.

EFFECT: higher effectiveness of the method.

5 cl, 1 dwg, 7 ex

FIELD: process engineering.

SUBSTANCE: invention relates to diamond grinding in making diamond rock cutting tool. Proposed method comprises processing the diamonds in velocity layer of magnetic fields together with ferromagnetic particles. Mix composed of ferromagnetic particles and diamond grains fills the cylindrical case by 0.25-0.35 of its volume. Diamond magnetic susceptibility is defined by the relationship: X1gR1(R1+R2)224μ0ρ2R22H2X2, where X1, X2 are diamond and ferromagnetic particle magnetic susceptibility, m3/kg; g is acceleration of gravity, m/s2; R1, R2 are diamond and ferromagnetic particle grain radii, m; µ0 is magnetic permeability of vacuum, (µ0=4π·107 GN/m); ρ2 is ferromagnetic particle density, kg/m3; H is magnetic field intensity, A/m. Note here that the relationship between diamond grain weight and that of ferromagnetic particles makes 0.51-0.61.

EFFECT: higher efficiency of grinding and quality of finished diamonds.

1 cl, 2 tbl, 1 ex

FIELD: process engineering.

SUBSTANCE: invention relates to production of carbon-based superhard composite to be used for making tools for mining, stone-working and metal working. Proposed method comprises applying high pressure and temperature to initial carbon component, a diamond, and binder. Note here that said carbon component comprises additionally fullerene and/or nanodiamond while said binder represents one or several components selected from the family including silicon bronze alloy, Monel metal, solid alloy. Superhard material is produced in two steps. At first step, the mix of initial components is subjected to dynamic pressure of 10-50 GPa at 900-2000°C. At second step, obtained material is placed in high-pressure vessel and subjected to static pressure of 5-15 GPa and heated to 700-1700°C for at least 20 seconds.

EFFECT: superhard micro hardness, high modulus of elasticity and higher wear resistance.

4 cl, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to colloidal chemistry and can be used in luminescent labels, as well as in production of materials for lasers, light diodes, solar panels, and photocatalysts. First, sodium sulfide and silver nitrate are prepared separately. For this purpose 0.01-0.5 g of sodium sulfide and 0.01-0.5 silver nitrate are dissolved in 40-200 ml of cold distilled water. 0.5-20 g of gelatin swell in reactor for 30 min in 100-500 ml of distilled water with temperature from 20-30°C. Obtained gelatin solution is heated to 40-90°C with mixing, 5 ml of 96% ethanol are poured into it. After that, double-stream pouring of prepared solutions of sodium sulfide and silver nitrate is realised, with further heating for 10-20 min with obtaining sol of colloidal silver sulfide quantum dots and cooling to 4-10°C for 10 hours. Obtained jelly is crushed to size of granules 5-10 mm, washed with distilled water at temperature 7-13°C, excess of water is decanted and granules are heated to temperature higher than 40°C.

EFFECT: invention makes it possible to obtain silver sulfide quantum dots with size 1-5 nm in gelatin matrix, luminescent in the range 800-1100 nm.

2 cl, 4 dwg, 2 ex

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