WO2012164008A1 - Method of evaluation of the effect of nanoparticles on cell cultures - Google Patents

Method of evaluation of the effect of nanoparticles on cell cultures Download PDF

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WO2012164008A1
WO2012164008A1 PCT/EP2012/060229 EP2012060229W WO2012164008A1 WO 2012164008 A1 WO2012164008 A1 WO 2012164008A1 EP 2012060229 W EP2012060229 W EP 2012060229W WO 2012164008 A1 WO2012164008 A1 WO 2012164008A1
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nanoparticles
cells
tested
cultures
determined
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PCT/EP2012/060229
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French (fr)
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WO2012164008A9 (en
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Ara Arshavirovich Abramyan
Viacheslav Ivanovich Beklemyshev
Liudmila Borisovna Buravkova
Konstantin Vitalievich Filippov
Anatoly Ivanovich Grigoriev
Umberto Orazio Giuseppe Maugeri
Vladimir Aleksandrovich Solodovnikov
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Closed Stock Company "Institute Of Applied Nanotechnology"
Fondazione Salvatore Maugeri Clinica Del Lavoro E Della Riabilitazione
Sib Laboratories Limited
Andreeva, Elena Romualdovna
Makhonin, Igor Ivanovich
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Publication of WO2012164008A1 publication Critical patent/WO2012164008A1/en
Publication of WO2012164008A9 publication Critical patent/WO2012164008A9/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention concerns biological research, namely the method of evaluation of the effect of nanoparticles on cell cultures.
  • mouse macrophage like cells 774.A1 mouse fibroblasts L929, embryonic mouse fibroblasts of the line Balb/3T3 (line 3T3), human monocytic lines THP-1 and HL-60 (peripheral blood, acute monocytic leukemia, promyelocytic leukemia), human lymphoid line K-562 (pleural fluid, chronic myelogenous leukemia) as models of cell cultures;
  • a method of estimating the effects of nanoparticles, in particular, gold (Au) with sizes ranging from 2 up to 200 nm on a T-cell lymphoblastic leukemia of a human being, of line Jurkat, based on the measurement of the level of gene expression is described in the technical specification of the application US N. 20100279289, pub. 04.11.2010.
  • a method is also known of estimating effects of nanoparticles on cell cultures (see application US N. 0080295187, pub. 11.27.2008) which consists in carrying out tests in vitro on cells of test-cultures containing suspensions of tested nanoparticles for determination of the effect of nanoparticles on the production of reactive oxygen species, formation of apoptotic and necrotic cells, disruption of mitochondrial function, using fluorescent cytological markers with subsequent analysis by flow cytometry; the method consists also in estimating the effects of nanoparticles on cells of test-culture by comparing the received indexes with corresponding indexes of control samples containing no testable nanoparticles.
  • DCFH-DA dichlorofluorescein diacetate
  • PI propidium iodide
  • DIOC6 3,3'- dihexyloxacarbocyanine iodide
  • ⁇ mitochondrial function
  • dye MitoSOXTM red
  • Macrophages like cells of mice RAW 264.7 were used as cells of test-cultures in experiments according to the given technical solution; thus according to the given invention it is described to use also epithelial and endothelial cells, cells of kidneys and liver, etc. as cells of test-cultures. They are isolated from invertebrates, mammals, bacteria and yeast.
  • ultra-dispersed particles of exhausts of energy (thermal) plants
  • nanoparticles of Carbon black nanoparticles of Ti0 2
  • fullerenes Fullerenes
  • micro-and nanospheres of polystyrene the mentioned nanoparticles are kept in cell cultures from 1 hour up to 4 weeks during their testing.
  • the effects of nanoparticles on cell cultures are determined according to the following indicators: expression of antioxidant molecules of glutathione (on the basis of identification of the induction of antioxidant enzymes, i.e. gems- oxygenase-1, superoxidedismutase, glutathioneperoxidase, glutathionereductase, catalase); activation of proinflammatory cascades during oxidative stress (on the basis of identification of the induction of proinflammatory cytokines and chemokines, for example, tumour-necrosis factor-a (TNF-a)); cellular absorption and intracellular localization of nanoparticles (on the basis of electron microscopy of macrophage like cells of mice RAW 264.7 and evaluation of changes (injuries) in mitochondria and vacuoles of cells).
  • antioxidant molecules of glutathione on the basis of identification of the induction of antioxidant enzymes, i.e. gems- oxygenase-1, superoxidedismutase, glutathioneperoxida
  • Tested cell cultures allow simulation of the basic functional features of the cellular system of a living organism.
  • each indicator for determination of the effect of nanoparticles on cells of test-cultures is researched using suspensions of nanoparticles with a concentration from 1- 10 - " 2 up to 1- 10 - " 4 % b.w.
  • Aqueous suspensions of nanoparticles at 1,0% b.w. concentration, and growth mediums in the ratios: 1: 100, 1: 1000, 1: 10000 are used for obtaining suspensions.
  • test-cultures are incubated with suspensions of tested nanoparticles, within 12- 36 hours.
  • a fluorescent marker dichlorodihydrofluorescein diacetate H 2 DCF-DA
  • determination of a number of stained cells and fluorescence intensity using the flow cytometer are used during in vitro tests for determination of effects of nanoparticles on the production of reactive oxygen species in cells of test-cultures.
  • a set of markers Annexin V with the analysis of tested cells using the flow cytometer is used for identification of apoptotic and necrotic cells in test-cultures.
  • the index of abnormalities in the mitochondria is identified by the change in mitochondrial transmembrane potential of cells of test-culture, using a fluorescent potential-dependent marker MitoTracker Red 580, by determination of fluorescence intensity with the help of the flow cytometer.
  • the index of cytotoxicity of tested nanoparticles, at exposure to multipotent mesenchymal stromal cells is evaluated by MTT-test method, using a solution of 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide, in a growth medium.
  • the index of cytotoxicity of tested nanoparticles at exposure to mononuclear cells - lymphocytes is evaluated from increase in the percentage of necrotic cells identified by staining with a marker - propidium iodide (PI) - and analysis in the flow cytometer.
  • PI propidium iodide
  • a fluorescent, pH-dependent, marker LysoTracker Green DND-26 with analysis of tested cells on the flow cytometer, is used for determination of the state of lysosomal compartment of tested cells.
  • measurement of lateral light scattering of cells of test- cultures using the flow cytometry is used for evaluation of intracellular accumulation of nanoparticles.
  • MMSC multipotent mesenchymal stromal cells
  • MNC mononuclear blood cells
  • MMSC are stromal progenitor cells (also called mesenchymal stemcells) are the principal cellular elements of the physiological tissue regeneration and tissue repair.
  • MNC are highly specialized differentiated cells that determine immune response against foreign influence.
  • MMSC and MNC represent populations that are spread throughout the body.
  • nanoparticles on MMSC and MNC nanoparticles of silicondioxide (Si0 2 ), nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag + ) or ions of cerium (Ce 3+ )) for indication of antimicrobial and antioxidant effects in manufactured products, which are used for medical, pharmaceutical, cosmetic and technical purposes.
  • the present invention can be realized by using known technological processes, as well as equipments and materials currently used in pharmacology and medicine.
  • Fig. 5 Evaluation of the viability of MNC (lymphocytes) after effect of nanoparticles.
  • Fig. 6 Evaluation of functional disorders of mitochondria of MNC(lymphocytes) after effect of nanoparticles.
  • Fig. 7 Evaluation of the production of reactive oxygen species (ROS) in MNC (lymphocytes) after effect of nanoparticles.
  • ROS reactive oxygen species
  • Fig. 8 Evaluation of the state of lysosomal compartment of MNC (lymphocytes) after effect of nanoparticles.
  • H 2 DCF-DA 2,7-dichlorodihydrofluorescein diacetate
  • Annexin V Annexin V
  • PI propidium-iodide
  • Nanoparticles used for realization of the invention are nanoparticles used for realization of the invention.
  • Nanoparticles of silicon dioxide Si0 2 .
  • Appearance it is a white powder with specific surface 300+30 m /g (DIN ISO 9277), hydrophilic; average size of the particles - 7 nm.
  • the initial 1.0% (10 mg/ml) aqueous suspension of nanoparticles of silicon dioxide (Si0 2 ) was prepared before the tests in vitro.
  • Sterile deionized water is used for the preparation of the aqueous suspension of nanoparticles.
  • Nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag + ) with a size of the particles of 20-200 nm; trade mark Moonclay ® .
  • Nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag + )are obtained using the method according to the patent RU N° 2330673 «Method of obtaining antimicrobial preparation* (pub.10.08.2008), developer and patentholder - CSC'Institute of Applied Nanotechnology” ( Russian).
  • the method of obtaining nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag + ) consists in modification of inorganic mineral with silicon-and aluminum- oxygen compounds, namely bentonite in Na-form, with inorganic salt of metal, in polar solvent.
  • bentonite is enriched with ions of Na + by its processing with 3- 10% aqueous solution of sodium chloride with subsequent washing and filtration of obtained semi-finished products.
  • Bentonite is modified with 10-20% solution of inorganic salt of metal; in this case silver nitrate (AgN0 3 ) is used.
  • Bentonite with a solution of silver nitrate (AgN0 3 ) is carried out at the following ratio, weight parts: bentonite : solution as l:(10-40).
  • Bentonite of Sarigyuh deposit (Armenia)is used as bentonite in Na-form.
  • Sterile deionized water is used for preparation of the aqueous suspension of nanoparticles.
  • Nanoparticles of bentonite intercalated by ions of cerium (Ce 3+ ) with a size of the particles of 20-200 nm.
  • Nanoparticles of bentonite (montmorillonite) intercalated by ions of cerium (Ce 3+ ) are obtained during modification of inorganic mineral with silicon and aluminum oxygen compounds, namely bentonite in Na-form with inorganic salt of metal in polar solvent.
  • bentonite is enriched with ions of Na + byits processing with 3- 10% aqueous solution of sodium chloride with the subsequent washing and filtration of obtained semi-finished products.
  • Bentonite is modified with a 10-20% solution of an inorganic salt of metal; in this case nitrate salt of cerium (Ce(N0 3 ) 3 -6H 2 0) is used.
  • Bentonite of Sarigyuh deposit (Armenia) is used as bentonite in Na-form.
  • the initial 1,0% suspensions of tested nanoparticles in deionized water specified by the present invention are the optimal conditions of formation of stable concentrated dispersion systems, required for evaluation of effects of nanoparticles on cells of test-cultures.
  • concentration of nanoparticles in deionized water At increase in concentration of nanoparticles in deionized water a process of deposition of nanoparticles is possible; at decrease in concentration of nanoparticles in deionized water, the subsequent process of evaluation of the biological effect on studied cells of test- cultures, becomes complicated.
  • aqueous suspensions of nanoparticles of silicon dioxide (Si0 2 ), nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag + ) and nanoparticles of bentonite (montmorillonite) intercalated by ions of cerium (Ce 3+ ), are added into corresponding growth mediums, for preparation of suspensions of tested nanoparticles.
  • suspensions of nanoparticles (used in the medium of test-cultures) determine most reliably effect of tested nanoparticles on the indexes of intracellular metabolism, as it is confirmed by description below.
  • MMSC multipotent mesenchymal stromal cells
  • MMSC i.e. stromal progenitor cells (also called mesenchymal stem cells) are the cellular elements of the physiological tissue regeneration and tissue repair. MMSC are substrate-dependent (adhesion) cell cultures.
  • MMSC was separated from stromal-vascular fraction of human adipose tissue (from the subcutaneous fat).
  • adipose tissue from the subcutaneous fat.
  • the fatty tissue of the blood was initially washed twice with phosphate buffer.
  • Adipose tissue was placed in a centrifuge tube for this purpose and the volume of the tube was increased with phosphate-buffered saline up to 50 ml so that the ratio of adipose tissue and buffer was 1:2.
  • the obtained mixture was centrifuged within 10 minutes, at acceleration 600 g (1000 rpm).
  • enzymatic disaggregation of the tissue was carried out.
  • the fatty tissue was weighed, a solution of collagenase IA was added up to a final concentration of 0,075% and was incubated within 30 minutes in a water bath at 37° C, with occasional shaking.
  • the enzyme was inactivated with an equal volume of complete growth medium containing 10% FBS, and was centrifuged within 10 minutes, at acceleration 600 g (1000 rpm).
  • a precipitate was obtained which was a stromal- vascular fraction of cells containing MMSC.
  • the precipitate was resuspended in growth medium (a-MEM) and passed through a 100-micron cell filters.
  • MMSC of the resulting suspension were obtained by their adhesion to plastic. To do this, the resulting cell suspension was placed in Petri dishes; planting density was
  • the obtained MMSC were placed in culture flasks with growth medium (a-MEM + 10% FBS) with the density 2- 10 4 cell/cm 2 and after reaching 70-80% confluence (degree of closure of a monolayer of cells) suspensions of the tested nanoparticles were injected into them.
  • growth medium a-MEM + 10% FBS
  • MNC mononuclear cells of blood
  • Lymphocytes are separated from human peripheral blood (healthy donors) by centrifugation.
  • the process of obtaining cultures of blood mononuclear cells (MNC) is carried out according to the Protocol of the company Amersham Biosciences (UK).
  • the blood collected in tubes containing 3,8 % sodium citrate was centrifuged within 25 minutes, at acceleration of 1800 g (3000 rpm). Blood plasma was discarded; phosphate buffer was added into the corpuscles, up to the original blood volume, and they were thoroughly resuspended. The obtained suspension was carefully layered on Ficoll Pague Plus at the ratio 2,5: 1 and centrifuged within 40 minutes, at 1800 g (3000 rpm). The interphase ring, represented by mononuclear cell fraction of blood, was collected and washed three times in phosphate buffer.
  • the number of cells was counted in the hematocytometer after separation of MNC, according to the protocol described above; then MNC were resuspended in growth medium RPMI-1640 + 5% FBS-taking into account lxlO 6 cells/ml and a suspension of researched nano particles was injected into them. The system was incubated within 24 hours.
  • MNC is a suspension culture, in which the cells are deposited quickly enough on the bottom of the culture flask.
  • the procedure for incubation of MNC with nanoparticles is carried out in the thermostat (37 °C) on a horizontal shaker, which provides continuous mixing of the medium.
  • Measurement of indicators selected for evaluation of the effect of nanoparticles on the state of cellular organelles is carried out by flow cytometry.
  • the method of flow cytometry is based on passing the cell suspension along a capillary, through the zone of sensitivity of the device; the speed of passage of cells through the capillary is about 1000 cells/sec. In the area of the device there is no more than one cell; the cells alternately cross the focused beam of light used to excite fluorescence.
  • the absorption and light scattering direct light scattering with angle 0,5-2° and angular (side) light scattering at the angle of 90°
  • cells as well as fluorescence of markers (dyes) associated with the cell are registered with the help of light-sensitive sensors (photodiodes and photomultipliers).
  • the flow cytometer Beckman Coulter Epics XL (USA) is used.
  • the obtained information is represented with frequency histograms, which are shown in the figures.
  • a number of living cells (under effects of nanoparticles) is estimated in the tests.
  • the principle of the MTT-test [Protocol of company Invitrogen (Molecular Probes), USA] is based on the ability of the enzyme of succinate d'ehydrogenase of the mitochondrial membrane of mammalian cells to restore the yellow salt 3-[4,5- dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT ) to formazan purple crystals, which are accumulated in the cytoplasm of living cells as a result of this reaction.
  • the level of mitochondrial respiration of cells which is an indicator of its viability, is determined according to the intensity of accumulation of crystals of formazan in the cytoplasm.
  • the amount of formazan formed in the cell monolayer is proportional to the current amount of living cells.
  • Formazan is extracted from cells with aprotic solvent DMSO - dimethylsulf oxide ((CH 3 ) 2 SO).
  • the intensity of the color of the solution is proportional to the amount of a live cells and is determined a colorimetric method.
  • a working solution of MTT (1,5 mg/ml 3-[4,5-dimethylthiazole-2-yl]-2,5- diphenyltetrazolium bromide) on the growth medium is prepared immediately before use.
  • Cells are incubated within 2 hours in a working solution of MTT in standard conditions (+37° C, 5% C0 2 ).
  • the reaction is stopped by removing the solution; then DMSO is added and the cells are placed on a shaker at 150 rpm for 15 minutes.
  • MTT-test method is designed to research the adhesion of cell cultures, so this method is used in the present invention for research of multipotent mesenchymal stromal cells (MMSC).
  • MNC mononuclear blood cells
  • lymphocytes i.e. lymphocytes
  • cytotoxicity is evaluated by detecting living MNC using labeling (staining) of cells with propidium iodide (PI). The results of staining MNC are estimated on the flow cytometer Beckman Coulter Epics XL (USA).
  • the samples were incubated within 1 hour in standard conditions (37° C, 5% C0 2 ), then washed three times with FBS and separated from plastic with a solution of trypsin-EDTA; the suspension was centrifuged for 10 min., at 500 g; deposit was resuspended in FBS and analyzed on the flow cytometer Beckman Coulter Epics XL (USA).
  • ROS reactive oxygen species
  • H 2 DCF-DA dichlorodihydrofluorescein diacetate
  • 10 mcg/ml a fluorescent marker - dichlorodihydrofluorescein diacetate
  • H 2 DCF-DA is a colorless ether which is transformed into a fluorescent product - H 2 DCF- when interacts with ROS. This product was visualized and the number of ROS in cells was estimated according to the intensity of its fluorescence.
  • H 2 DCF-DA was added into the cell cultivated medium for 30 minutes, before determining the level of ROS in researches of intracellular level of ROS; then cells were washed twice with a medium with serum.
  • MNC Mononuclear cells of blood
  • MMSC Multipotent mesenchymal stromal cells
  • Suspensions were centrifuged within 10 min., at acceleration 500 g; the deposit was resuspended in phosphate buffer.
  • ROS reactive oxygen species
  • compartmentalization of cells suggests that biochemical processes in cells are localized in certain compartments. Most of the organelles in cells are the compartments including lysosomes.
  • Cells were incubated with LysoTracker within 1 hour in standard conditions (37°C, 5% C0 2 ) and analyzed on the flow cytometer Beckman Coulter Epics XL (USA).
  • Annexin V is used for evaluation of effects of nanoparticles on the cells. It allows detection both of apoptotic and necrotic cells, according to the Protocol of Immunotech, France. In the presence of Ca 2+ and Mg 2+ , Annexin V interacts with phosphatidylserine which is shifted from the inner cell membrane to the outside one in the early stages of apoptosis. The cell membrane of living cells is impermeable to propidium iodide (PI).
  • PI propidium iodide
  • propidium iodide passes through the cell membrane and interacts with the minor groove of deoxyribonucleic acid(DNA). Double staining with Annexin V and propidium iodide indicates late stages of apoptosis.
  • Cells were washed with cold phosphate-buffered saline (PBS) and incubated in a solution within 15 min. at +4° C or on ice, without access of light. Then the cells were washed with growth medium and analyzed in the fluorescent phase-contrast microscope Leica DM5000B (filter BP 450-490, LP 520 for Annexin V; filter BP 510-560, LP 590 filter for propidium iodide).
  • the cells are separated from the plastic with a solution of 0,05% trypsin - 0,04% EDTA, and washed three times with PBS; then cell suspensions are stained. The results of staining are estimated by the flow cytometer Beckman Coulter Epics XL (USA). The amount of live cells, necrotic and apoptotic cells (ratio of necrotic and apoptotic ways of cell death) after the effects of nanoparticles were determined by flow cytometry.
  • Intracellular accumulation of nanoparticles is detected by the appearance of cells with a modified side light scatter, using the flow cytometer Beckman Coulter Epics XL (USA), highly sensitive sensors of the cytometer located near the flow cell, fix the light scattering angle from 2 up to 19° (it is called direct or small-angle light scattering (FSC)) and the angle of 90°, i.e. side light scattering (SSC).
  • FSC direct or small-angle light scattering
  • SSC side light scattering
  • SSC side scatter
  • Example 1 In culture flasks (5 ml of growth medium in each) there are MMSC test-cultures and suspensions of nanoparticles of Si0 2 , at concentration of nanoparticles in suspensions of 1-10 "2 ; 1-10 "3 ; 1 ⁇ 10 "4 % b.w..
  • Example 2 In culture flasks (5 ml of growth medium in each) there are MMSC test-cultures and suspensions of nanoparticles of bentonite intercalated by ions of cerium (Ce 3+ ), at concentration of nanoparticles in suspensions of 1-10 "2 ; 1-10 "3 ; 1 ⁇ 10 "4 % b.w..
  • Example 3 In culture flasks (5 ml of growth medium in each) there are MMSC test-cultures and suspensions of nanoparticles of bentonite intercalated by ions of silver(Ag + ), at concentration of nanoparticles in suspensions of 1-10 "2 ; 1-10 "3 ; 1 ⁇ 10 "4 % b.w.. Measurements were carried out on samples according to Examples 4-6 for MNC test-cultures.
  • Example 4 In culture flasks (5 ml of growth medium in each) there are MNC test- culture and suspensions of nanoparticles of Si0 2 at concentration of nanoparticles insuspensions: MO "2 ; MO "3 ; l-10 ⁇ 4 b.w. .
  • Example 5.1n culture flasks (5 ml of growth medium in each) there are MNC test- cultures and suspensions of nanoparticles of bentonite intercalated by ions of cerium (Ce 3+ ), at concentration of nanoparticles in suspensions of 1-10 "2 ; 1-10 "3 ; l-10 ⁇ 4 b.w. %.
  • Example 6 In culture flasks (5 ml of growth medium in each) there are MNC test- cultures and suspensions of nanoparticles of bentonite intercalated by ions of silver (Ag + ), at concentration of nanoparticles in suspensions of 1-10 - " 2 ; 1-10 - " 3 ; 1-10 -4 b.w. .
  • Control samples of primary cultures of multipotent mesenchymal stromal cells (MMSC) and mononuclear cells (MNC) -lymphocytes, according of Examples 7 and 8 are used for carrying out researches.
  • MMSC multipotent mesenchymal stromal cells
  • MNC mononuclear cells
  • Example 7 In the culture flask there is MMSC test-culture (without nanoparticles), growth medium - 4,95 ml and deionized water - 50 ml.
  • Example 8 In the culture flask there is MNC test-culture (without nanoparticles), growth medium - 4,95 ml and deionized water - 50 ml.
  • MMSC primary multipotent mesenchymal stromal cells
  • MNC mononuclear cells
  • Example 1 It was revealed by the researches that the nanoparticles according to Example 1 did not affect the amount of MMSC in which mitochondria (cells stained with MitoTracker, %) were detected. However, at the concentration of nanoparticles of 1-10 " % b.w. in Example 1, there was a significant decrease in the average intensity of intracellular fluorescence of the marker (MitoTracker/cells, conv. units). That indicates a decrease in mitochondrial transmembrane potential ( ⁇ ) of tested cells.
  • ROS reactive oxygen species
  • Example 1-3 the share of MMSC (cells stained with H 2 DCF-DA, %) with detected reactive oxygen species (ROS) was small and did not exceed 3%.
  • Example 1 the share of MMSC (cells stained with H 2 DCF-DA, %) (in which reactive oxygen species (ROS) were detected) was greater at the concentration of nanoparticles of 1-10 " % b.w. (in comparison with Examples 2 and 3).
  • ROS reactive oxygen species
  • Intracellular accumulation of nanoparticles is diagnosed owing to appearance of cells with changed (increased) light scattering.
  • tested nanoparticles according to Examples 2 and 3, at the concentration 1-10 " , show a share of cells with changed light scattering of less than 15% of the tested cells.
  • the average intensity of intracellular fluorescence of the marker (MitoTracker/cells, conv. units), indicating a change (decrease) of the transmembrane potential of mitochondria(A v Pm), varied considerably depending on the type and concentration of the nanoparticles (Examples 4, 5 and 6).
  • ROS reactive oxygen species
  • ROS reactive oxygen species
  • Example 6 It was revealed by the search that, according to Example 6, decrease of ROS in cells (in comparison with control) was detected at the concentration of nanoparticles of 1-10 " % b.w.. At low concentrations, the level of ROS roughly corresponded to the level of ROS in control (without nanoparticles) (Example 8).
  • Example 8 The average intensity of intracellular fluorescence of the lysosomal marker (LysoTracker/cells, conv. units), which indicates activity of lysosomes, varied insignificantly with respect to the values in the control (Example 8), at all concentrations of nanoparticles tested. However, there is increased activity of lysosomes in Example 4 and a decrease in their activity according to Example 6, at concentration of nanoparticles l-10 "2 % b.w..
  • Nanoparticles according to Example 4 did not provide considerable effect on the share of apoptotic cells, which was in the range 15,1 - 16,5 %.
  • Intracellular accumulation of nanoparticles is revealed by the appearance of cells with changed (increased) light scattering. It was revealed by the search that tested nanoparticles according to Example 4, at concentration 1- 10 - " 2 ; 1-10 - " 3 ;1-10 - ⁇ 4 % b.w. do not cause a considerable increase in light scattering of cells. Amount of cells with intact light scattering was in the range:88,8 - 90,2%; for the control example 8 (without nanoparticles) - 89,1%.
  • lymphocytes Primary cultures of multipotent mesenchymal stromal cells and mononuclear blood cells (lymphocytes) are used as cells of test-cultures. They are principal cellular elements determining the physiological tissue regeneration and repair, immune response against external stress. These important cell populations allow simulating elements of the cell system of a human being and comprehensively assessing potential biomedical risk of exposure to nanoparticles at the level of the whole organism.
  • nanoparticles of silicon dioxide (Si0 2 ), nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag + ) and nanoparticles of bentonite (montmorillonite) intercalated by ions of cerium (Ce 3+ ) are perspective nanomaterials for medical, pharmacological, cosmetic ant technical purposes.
  • Tested nanoparticles allow evaluating various levels of their biological effect on cells of test-cultures, due to selected indicators of cell activity.
  • the methods developed for evaluation in vitro of biological effects of tested nanoparticles on cells of test-cultures allow reliably estimation of the effect of the type of nanoparticles and their concentration (concentration effect) on indicators of cell activity and allow to take a decision about the possible risks of using biomedical nanoparticles.

Abstract

A method is described to evaluate in vitro the biological effect of bactericidal inorganic nanoparticles on the cellular system of a living organism, with the aim to state the possibility of using them in the pharmaceutical, medical, cosmetic industry The method has essentially identified a set of biologic indexes, which must be determined on primary cells cultures of multipotent mesenchymal stromal cells (also called mesenchymal stem cells, MSC) separated from stromal-vascular fraction of human adipose tissue and on mononuclear cells-lymphocytes separated from human peripheral blood, both cultures containing the bactericidal inorganic nanoparticles. The indexes (such as cell viability (cytotoxicity), function of mitochondria, production of reactive oxygen species (ROS), state of lysosomal compartment, necrosis and apoptosis, intracellular accumulation of nanoparticles) are evaluated comprehensively, in comparison with the corresponding indexes obtained in the absence of the nanoparticles in the cells cultures.

Description

METHOD OF EVALUATION OF THE EFFECT OF NANOP ARTICLES ON CELL CULTURES
Field of the invention
The present invention concerns biological research, namely the method of evaluation of the effect of nanoparticles on cell cultures.
Rapidly developing researches of properties of nanomaterials and nanoparticles and permanently expanding application areas put the problem of finding and testing methods to assess their effects on living organisms, both at the level of the whole organism and of separate cells and cellular structures. Evaluation of cellular effects is particularly important owing to nano dimension of active agents that directly affect cellular structures. Their interaction with the cellular system of different tissues and organs takes place regardless of the way in which nanoparticles and nanomaterials enter the body (for example when applied to the skin or by injection into the tissues or blood). It affects the functioning of cellular organelles and cell viability and may have a toxic effect on them.
Therefore, the problem of creating tests for evaluation of the biological activity of nanoparticles and their effects on cell cultures, taking into account physical, chemical and biological features of both nanoparticles and cell cultures, is urgent and important.
State of the art
The methods (tests) in vitro to determine the effect of nanoparticles on cell cultures are known. According to them it is recommended to use the following procedures and materials:
- mouse macrophage like cells 774.A1, mouse fibroblasts L929, embryonic mouse fibroblasts of the line Balb/3T3 (line 3T3), human monocytic lines THP-1 and HL-60 (peripheral blood, acute monocytic leukemia, promyelocytic leukemia), human lymphoid line K-562 (pleural fluid, chronic myelogenous leukemia) as models of cell cultures;
- tested nanoparticles dispersed in a solvent for injection into the corresponding nutrient medium of cell culture;
- carrying out tests in vitro for evaluation of effects of nanoparticles on cell cultures according to the following terms: cytotoxicity, production of reactive oxygen species (ROS), production of factor necrosis of tumor-alpha, immunotropic effect by using in particular the method of flow cytometry for both qualitative and quantitative changes in cells (see Methodological Guidelines MU 1.2.2635-10 "Medical-biological evaluation of the safety of nanomaterials." Approved by the Chief Medical Officer of the Russian Federation 24.05.2010).
A method of estimating the level of toxicity, stress response, DNA damage of epithelial cells, normal human keratinocytes (NHK), human fibroblasts (HSF) under the influence of multi-layered carbon nano tubes with diameters ranging from 10 up to 50 nm which are used, in particular, with chemical-therapeutic purposes in oncology, was described in the technical specification of the application US N. 20090269279, pub. 29.10.2009). The tests indicated in said technical document for measurement of cell proliferation and of the degree of apoptosis and necrosis, are used for evaluation of the effect of mentioned nanomaterials on cell cultures, using the method of cytometry.
A method of estimating the effects of nanoparticles, in particular, gold (Au) with sizes ranging from 2 up to 200 nm on a T-cell lymphoblastic leukemia of a human being, of line Jurkat, based on the measurement of the level of gene expression is described in the technical specification of the application US N. 20100279289, pub. 04.11.2010.
A method is also known of estimating effects of nanoparticles on cell cultures (see application US N. 0080295187, pub. 11.27.2008) which consists in carrying out tests in vitro on cells of test-cultures containing suspensions of tested nanoparticles for determination of the effect of nanoparticles on the production of reactive oxygen species, formation of apoptotic and necrotic cells, disruption of mitochondrial function, using fluorescent cytological markers with subsequent analysis by flow cytometry; the method consists also in estimating the effects of nanoparticles on cells of test-culture by comparing the received indexes with corresponding indexes of control samples containing no testable nanoparticles.
This technical solution is selected as the closest prior art for the present invention.
In the specified technical solution the following standard cytology dyes are used as fluorescent cytological markers, added to cells for visualization and control of functional changes in cell structures: dichlorofluorescein diacetate (DCFH-DA) - to detect ROS, propidium iodide (PI) - to identify apoptotic and necrotic cells, 3,3'- dihexyloxacarbocyanine iodide (DiOC6) - to detect violations of mitochondrial function (changes of transmembrane potential in mitochondria (ΔΨιη)); dye MitoSOX™ (red) - to detect ROS and functional changes in cells. Macrophages like cells of mice RAW 264.7 were used as cells of test-cultures in experiments according to the given technical solution; thus according to the given invention it is described to use also epithelial and endothelial cells, cells of kidneys and liver, etc. as cells of test-cultures. They are isolated from invertebrates, mammals, bacteria and yeast.
The following materials are used as test-nanoparticles in determining in vitro the effects of nanoparticles on cell cultures: ultra-dispersed particles (UFP) of exhausts of energy (thermal) plants, nanoparticles of Carbon black, nanoparticles of Ti02, fullerenes (Fullerol), micro-and nanospheres of polystyrene; the mentioned nanoparticles are kept in cell cultures from 1 hour up to 4 weeks during their testing.
In particular, macrophage like cells of mice of test-culture RAW 264.7, without injection of nanoparticles, were used as control sample.
In this technical solution the effects of nanoparticles on cell cultures are determined according to the following indicators: expression of antioxidant molecules of glutathione (on the basis of identification of the induction of antioxidant enzymes, i.e. gems- oxygenase-1, superoxidedismutase, glutathioneperoxidase, glutathionereductase, catalase); activation of proinflammatory cascades during oxidative stress (on the basis of identification of the induction of proinflammatory cytokines and chemokines, for example, tumour-necrosis factor-a (TNF-a)); cellular absorption and intracellular localization of nanoparticles (on the basis of electron microscopy of macrophage like cells of mice RAW 264.7 and evaluation of changes (injuries) in mitochondria and vacuoles of cells).
It is possible to draw the following conclusions on the basis of the analysis of the above mentioned technical solutions:
- there is no system of tests capable to evaluate biological activity, toxicity and safety of existing nanomaterials and nanoparticles in relation to cellular cultures;
- developpers generally offer highly specialized and insufficiently standardized methodologies and tests;
- certain cultures of cells (biological liquids, tissues and bodies of living organisms) and concrete kinds of nanomaterials and nanoparticles are tested in the above mentioned technical solutions with the purpose of their particular application in the field of biology, medicine or technics; - some of the major types of primary cultures of cells, responsible for physiological tissue regeneration and tissue repair after injury, and determining the immune response against external effects, were not tested in the above mentioned technical solutions;
- in the above technical solutions the effects of nanoparticles on the major cell organelles - lysosomes (they are responsible for digestion of substances or particles trapped with cells at endocytosis and for autophagy and autolysis) were not tested and the effects of nanoparticles on the functioning cellular organelles in general depending on their concentration in cell medium were also not tested.
Summary of the invention.
From the standpoint of modern biological researches and nanotechnology it is important to resolve the technical problem of the expansion, improvement of the system of tests (methods) for the determination of the biological effects of nanoparticles on tested cell cultures, based on their physical-chemical and bio-active properties. Tested cell cultures allow simulation of the basic functional features of the cellular system of a living organism.
To resolve this technical problem it is described a method for evaluation of the effects of nanoparticles on cell cultures which comprises the following steps:
- carrying out tests in vitro on cells of test-cultures containing a suspension of tested nanoparticles;
- determination of the effect of nanoparticles on the production of reactive oxygen species;
- formation of apoptotic and necrotic cells;
- disruption of mitochondrial function by using cytological fluorescent markers, end subsequent analysis by the flow cytometry;
- evaluation of effects of nanoparticles on tested cells of the test-culture, by comparing the obtained indexes with the corresponding indexes of the control samples of tested cells of the test-culture, not containing tested nanoparticles.
Primary cultures of multipotent mesenchymal stromal cells, separated from stromal- vascular fraction of human adipose tissue and mononuclear cells - lymphocytes separated from human peripheral blood, are used as cells of test-cultures. The effect of nanoparticles on the named cells of test-cultures, in so far as cytotoxicity of lysosomal compartments and intracellular accumulation of the tested nanoparticles are concerned, is also determined. For carrying out tests in vitro, each indicator of the effect of nanoparticles on cells of test- cultures is determined using suspensions of tested nanoparticles and changing their concentration in the suspension. Each index of test in vitro is compared with the corresponding index of test in vitro of the control sample, depending on the concentration of nanoparticles. The effect of nanoparticles on vital activity of cells is evaluated as a whole according to the obtained values.
According to the present invention, during in vitro testing, each indicator for determination of the effect of nanoparticles on cells of test-cultures is researched using suspensions of nanoparticles with a concentration from 1- 10 -"2 up to 1- 10 -"4 % b.w. Aqueous suspensions of nanoparticles at 1,0% b.w. concentration, and growth mediums in the ratios: 1: 100, 1: 1000, 1: 10000 are used for obtaining suspensions.
According to the present invention silicon dioxide with a size of the particles of 5- 12 nm, nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag+) with a size of the particles of 20-200 nm, nanoparticles of bentonite (montmorillonite) intercalated by ions of cerium (Ce3+) with a size of the particles of 20-200 nm, are used as tested nanoparticles.
According to the present invention test-cultures are incubated with suspensions of tested nanoparticles, within 12- 36 hours.
According to the present invention, a fluorescent marker dichlorodihydrofluorescein diacetate (H2DCF-DA)-, and determination of a number of stained cells and fluorescence intensity using the flow cytometer, are used during in vitro tests for determination of effects of nanoparticles on the production of reactive oxygen species in cells of test-cultures.
According to the present inventiona a set of markers Annexin V with the analysis of tested cells using the flow cytometer, is used for identification of apoptotic and necrotic cells in test-cultures.
According to the present invention the index of abnormalities in the mitochondria is identified by the change in mitochondrial transmembrane potential of cells of test-culture, using a fluorescent potential-dependent marker MitoTracker Red 580, by determination of fluorescence intensity with the help of the flow cytometer.
According to the present invention the index of cytotoxicity of tested nanoparticles, at exposure to multipotent mesenchymal stromal cells, is evaluated by MTT-test method, using a solution of 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide, in a growth medium.
According to the present invention the index of cytotoxicity of tested nanoparticles at exposure to mononuclear cells - lymphocytes, is evaluated from increase in the percentage of necrotic cells identified by staining with a marker - propidium iodide (PI) - and analysis in the flow cytometer.
According to the present invention a fluorescent, pH-dependent, marker LysoTracker Green DND-26, with analysis of tested cells on the flow cytometer, is used for determination of the state of lysosomal compartment of tested cells.
According to the present invention, measurement of lateral light scattering of cells of test- cultures using the flow cytometry is used for evaluation of intracellular accumulation of nanoparticles.
By realization of the present invention the effective method of evaluation in vitro of the biological effect of nanoparticles (which are perspective for medical and technical application) on cellular cultures, which simulate the basic functional features of cellular system of a living organism, was developed. The importance of the new method is explained as follows:
- integrated assessment of the biological effect of nanoparticles on such important types of cells as the primary culture of multipotent mesenchymal stromal cells (MMSC) and mononuclear blood cells (MNC) -lymphocytes; MMSC are stromal progenitor cells (also called mesenchymal stemcells) are the principal cellular elements of the physiological tissue regeneration and tissue repair. MNC are highly specialized differentiated cells that determine immune response against foreign influence. MMSC and MNC represent populations that are spread throughout the body. Thus, the evaluation in vitro of effects of nano particles on the cells of tissue systems of the internal medium will allow describing the possible biomedical risks of use of nanoparticles at the level of the whole organism;
- reasonability of developing a method for determination of the biological effect of nanoparticles on MMSC and MNC by assessing in vitro the full range of parameters that characterize the indexes of cell life: viability and ways of cell death, the energy potential and the system of degradation of endocited products (cytotoxicity, production of reactive oxygen species (ROS); necrotic and apoptotic ways of cell death, disruption of mitochondria, change of lysosomal compartments, intracellular accumulation of nanoparticles);
- reasonability of determination of the biological effect of nanoparticles on MMSC and MNC (nanoparticles of silicondioxide (Si02), nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag+) or ions of cerium (Ce3+)) for indication of antimicrobial and antioxidant effects in manufactured products, which are used for medical, pharmaceutical, cosmetic and technical purposes.
Analysis of available art did not reveal technical solutions with a set of characteristics corresponding to those of the present declared technical solution and able to realize the above described results.
The analysis of available art testifies the conformity of the present technical solution to criteria of "novelty" and "degree of inventiveness".
The present invention can be realized by using known technological processes, as well as equipments and materials currently used in pharmacology and medicine.
Brief description of the drawings.
Fig. l. Evaluation of viability of MMSC after effect of nanoparticles.
Fig.2. Evaluation of functional disorders of mitochondria of MMSC after effect of nanoparticles.
Fig.3. Evaluation of the production of reactive oxygen species (ROS) in MMSC after effect of nanoparticles.
Fig.4. Evaluation of the state of lysosomal compartment of MMSC after effect of nanoparticles.
Fig. 5. Evaluation of the viability of MNC (lymphocytes) after effect of nanoparticles.
Fig. 6. Evaluation of functional disorders of mitochondria of MNC(lymphocytes) after effect of nanoparticles.
Fig. 7. Evaluation of the production of reactive oxygen species (ROS) in MNC (lymphocytes) after effect of nanoparticles.
Fig. 8. Evaluation of the state of lysosomal compartment of MNC (lymphocytes) after effect of nanoparticles.
Detailed description of the invention.
The following devices and equipments used in biotechnologies were used for realization of the invention: a laminar case (VL22, Sampo, Russia); the C02-incubator (Sanyo, Japan); a light phase-contrast inverted microscope Leica DM IL (Germany); a fluorescent phase-contrast microscope Leica DM5000B (Germany) equipped with the mercury lamp HBO 100 AC; sets of filters for the analysis of fluorescence UV (BP 340- 380, LP 425), UV/V (BP 355-425, LP 470), FITC (BP 450-490, LP 515), TRITC (BP 515- 560, LP 590); the chamber and the system of analysis of image DC420 (Leica, Germany); a shaker Vortex (Elmi, Latvia); a centrifuge Eppendorf 5204 R (Eppendorf, Germany); a water bath (BioSan); a flow cytometer Coulter Epics XL (Beckman Coulter, USA); automatic pipettes (Eppendorf, Germany); hematocytometer (Bright Line, USA); a refrigerator (Indesit, Russia) and a low-temperature refrigerator (Sanyo, Japan).
The following materials and substances were used for realization of the invention:
- fluorescent marker - 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) (company Invitrogen GmbH, Germany);
- set of markers Annexin V (Annexin V-FITC, propidium-iodide (PI), binding buffer) [company Immunotech, France, company Invitrogen (Molecular Probes), USA];
- fluorescent potential-dependent marker Mito Tracker Red 580 [company Invitrogen (MolecularProbes), USA; company Invitrogen GmbH, Germany];
reagent of MTT-test - 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide[company ICN Pharmaceutical, USA];
- fluorescent marker - propidium iodide, (PI) [company Sigma, USA] ;
- fluorescent pH-dependent marker LysoTracker GreenDND-26 [company Invitrogen GmbH, Germany] ;
- aprotic solvent- dimethylsulfoxide (CH3)2SO [company «Biolot», Russia];
- fetal bull serum(FBS) [company HyClone, USA; PromoCell, Germany];
- growth (nutrient) medium a - MEM [company ICN Pharmaceutical, USA] (for culture of MMSC) ;
- growth (nutrient) medium RPMI-1640 (Institute of Poliomyelitis and Viral Encephalitis named
M.P. Chumakov of RAS, Russia) (for culture of MNC);
- phosphate-buffered saline(PBS) [company Gibco, United Kingdom];
- solution: Trypsin -EDTA [company Gibco, United Kingdom];
- collagenase IA [company Sigma- Aldrich, USA] ; - density gradient Ficoll Histopaque [companySigma, USA] ;
- deionized water [company «SigmaTeck», Russia].
Nanoparticles used for realization of the invention:
Nanoparticles of silicon dioxide (Si02).
Appearance: it is a white powder with specific surface 300+30 m /g (DIN ISO 9277), hydrophilic; average size of the particles - 7 nm. Trade mark of the product- AEROSIL® 30;manufacturer - Evonik Degussa GmbH, Germany.
The initial 1.0% (10 mg/ml) aqueous suspension of nanoparticles of silicon dioxide (Si02) was prepared before the tests in vitro. Sterile deionized water is used for the preparation of the aqueous suspension of nanoparticles.
- Nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag+) with a size of the particles of 20-200 nm; trade mark Moonclay®.
Nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag+)are obtained using the method according to the patent RU N° 2330673 «Method of obtaining antimicrobial preparation* (pub.10.08.2008), developer and patentholder - CSC'Institute of Applied Nanotechnology" (Russia).
The method of obtaining nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag+) consists in modification of inorganic mineral with silicon-and aluminum- oxygen compounds, namely bentonite in Na-form, with inorganic salt of metal, in polar solvent.
Before modification bentonite is enriched with ions of Na+by its processing with 3- 10% aqueous solution of sodium chloride with subsequent washing and filtration of obtained semi-finished products.
Bentonite is modified with 10-20% solution of inorganic salt of metal; in this case silver nitrate (AgN03) is used.
Incubation of modified bentonite in the mentioned salt solution is carried out within 12-24 hours; then bentonite is cleaned from salts of sodium by washing and filtration; after drying (at a temperature not higher than 100°C)the obtained product is grinded up to a dispersiveness of the particles of 20-150 nm.
Processing of bentonite with a solution of silver nitrate (AgN03) is carried out at the following ratio, weight parts: bentonite : solution as l:(10-40). Bentonite of Sarigyuh deposit (Armenia)is used as bentonite in Na-form.
Water or aqueous-alcohol solutions are used as polar solvent.
The initial 1,0 % (10 mg/ml) aqueous suspension of nanoparticles of bentonite (montmorillonite), intercalated by ions of silver (Ag+), is prepared before carrying out the tests in vitro. Sterile deionized water is used for preparation of the aqueous suspension of nanoparticles.
- Nanoparticles of bentonite (montmorillonite) intercalated by ions of cerium (Ce3+) with a size of the particles of 20-200 nm.
Nanoparticles of bentonite (montmorillonite) intercalated by ions of cerium (Ce3+) are obtained during modification of inorganic mineral with silicon and aluminum oxygen compounds, namely bentonite in Na-form with inorganic salt of metal in polar solvent.
Before modification bentonite is enriched with ions of Na+byits processing with 3- 10% aqueous solution of sodium chloride with the subsequent washing and filtration of obtained semi-finished products.
Bentonite is modified with a 10-20% solution of an inorganic salt of metal; in this case nitrate salt of cerium (Ce(N03)3-6H20) is used.
Incubation of modified bentonite in the mentioned salt solution is carried out within 12-24 hours; then bentonite is cleaned from salts of sodium by washing and filtration and after drying (at a temperature not higher then 100°C), the obtained product is grinded up to dispersiveness of particles of 20-150 nm.
Processing bentonite with solution of nitrate salt of cerium (Ce(N03)3-6H20) is carried out at the following ratio, weight parts: bentonite : solution as l:(10-40).
Bentonite of Sarigyuh deposit (Armenia) is used as bentonite in Na-form.
Water or aqueous-alcohol solutions are used as polar solvent.
Initial 1,0 % (10 mg/ml) aqueous suspension of nanoparticles of bentonite (montmorillonite) intercalated by ions of cerium (Ce3+) is prepared before carrying out tests in vitro. Sterile deionized water is used for preparation of the aqueous suspension of nanoparticles.
The initial 1,0% suspensions of tested nanoparticles in deionized water specified by the present invention are the optimal conditions of formation of stable concentrated dispersion systems, required for evaluation of effects of nanoparticles on cells of test-cultures. At increase in concentration of nanoparticles in deionized water a process of deposition of nanoparticles is possible; at decrease in concentration of nanoparticles in deionized water, the subsequent process of evaluation of the biological effect on studied cells of test- cultures, becomes complicated.
For tests in vitro 1,0% aqueous suspensions of nanoparticles of silicon dioxide (Si02), nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag+) and nanoparticles of bentonite (montmorillonite) intercalated by ions of cerium (Ce3+), are added into corresponding growth mediums, for preparation of suspensions of tested nanoparticles.
Initial 1,0% aqueous suspensions of nanoparticles and growth mediums (a-MEM for culture ofMMSC and RPMI-1640 for culture ofMNC) at a ratio: 1: 100; 1: 1000; 1: 10000 are used for preparation of suspensions of nanoparticles with a concentration from 1· 10" up to 1- 10"4% by weight (b.w.).
The following suspensions are specifically used, to obtain a suspension of nanoparticles (per 10 ml):
- at the concentration of nanoparticles 1- 10" % b.w.: growth medium - 9,9 ml; 1,0 % aqueous suspension of nanoparticles- 0,1 ml;
- at the concentration of nanoparticles 1- 10" % b.w.: growth medium - 9,99 ml ; 1,0 % aqueous suspension of nanoparticles - 0, 01 ml;
- at the concentration of nanoparticles 1· 10"4% b.w.: growth medium - 9,999 ml; 1,0 % aqueous suspension of nanoparticles - 0, 001 ml.
At the indicated concentrations, suspensions of nanoparticles (used in the medium of test-cultures) determine most reliably effect of tested nanoparticles on the indexes of intracellular metabolism, as it is confirmed by description below.
Cell sof test-cultures are used for realization of the invention.
Initial cultures of multipotent mesenchymal stromal cells (MMSC).
MMSC, i.e. stromal progenitor cells (also called mesenchymal stem cells) are the cellular elements of the physiological tissue regeneration and tissue repair. MMSC are substrate-dependent (adhesion) cell cultures.
MMSC was separated from stromal-vascular fraction of human adipose tissue (from the subcutaneous fat). For obtaining the primary culture of MMSC it was used the method described in the patent RU N. 2351649 "Method of obtaining cultures of human mesenchymal stromal cells separated from lipoaspirat" (pub. 10.04.2009); developer and patent holder -the Institute for Biomedical Problems and RAS and methodology described in the work of Zuk PA, Zhu M., Mizuno H. et al. "Human adipose tissue is a source of multipotent stem cells". Molecular biology of the cell. 2002, Vol.13, pp. 4279-4295.
The fatty tissue of the blood was initially washed twice with phosphate buffer. Adipose tissue was placed in a centrifuge tube for this purpose and the volume of the tube was increased with phosphate-buffered saline up to 50 ml so that the ratio of adipose tissue and buffer was 1:2. The obtained mixture was centrifuged within 10 minutes, at acceleration 600 g (1000 rpm). Then enzymatic disaggregation of the tissue was carried out. To do this, after washing, the fatty tissue was weighed, a solution of collagenase IA was added up to a final concentration of 0,075% and was incubated within 30 minutes in a water bath at 37° C, with occasional shaking. Then the enzyme was inactivated with an equal volume of complete growth medium containing 10% FBS, and was centrifuged within 10 minutes, at acceleration 600 g (1000 rpm).
A precipitate was obtained which was a stromal- vascular fraction of cells containing MMSC. The precipitate was resuspended in growth medium (a-MEM) and passed through a 100-micron cell filters. MMSC of the resulting suspension were obtained by their adhesion to plastic. To do this, the resulting cell suspension was placed in Petri dishes; planting density was
3Ί0 3 cell/cm 2. Not adherent cells were removed after 24 hours; primary cultures were washed with PBS and fresh complete growth medium (a-MEM + 10% FBS) was added. After reaching 80-90% confluence (degree of closure of a monolayer of cells), MMSC were decanted. Cells obtained after 2-4 decanting were used in researches.
When subcultivating, the obtained MMSC were placed in culture flasks with growth medium (a-MEM + 10% FBS) with the density 2- 104cell/cm2 and after reaching 70-80% confluence (degree of closure of a monolayer of cells) suspensions of the tested nanoparticles were injected into them.
Cells without nanoparticles, incubated under the same conditions, were used for determination of initial (control) values of the researched parameters.
Samples for analysis of researched indexes were prepared after 24 hours of incubation (see below).
Initial cultures of mononuclear cells of blood (MNC) - lymphocytes. MNC are differentiated cells that determine immune response against alien impact. MNC are the suspension of culture of cells.
Lymphocytes (leukocytes) are separated from human peripheral blood (healthy donors) by centrifugation. The process of obtaining cultures of blood mononuclear cells (MNC) is carried out according to the Protocol of the company Amersham Biosciences (UK).
For this purpose, the blood collected in tubes containing 3,8 % sodium citrate was centrifuged within 25 minutes, at acceleration of 1800 g (3000 rpm). Blood plasma was discarded; phosphate buffer was added into the corpuscles, up to the original blood volume, and they were thoroughly resuspended. The obtained suspension was carefully layered on Ficoll Pague Plus at the ratio 2,5: 1 and centrifuged within 40 minutes, at 1800 g (3000 rpm). The interphase ring, represented by mononuclear cell fraction of blood, was collected and washed three times in phosphate buffer.
The number of cells was counted in the hematocytometer after separation of MNC, according to the protocol described above; then MNC were resuspended in growth medium RPMI-1640 + 5% FBS-taking into account lxlO6 cells/ml and a suspension of researched nano particles was injected into them. The system was incubated within 24 hours.
MNC is a suspension culture, in which the cells are deposited quickly enough on the bottom of the culture flask. In this regard, in order to improve the contact of cells with the tested nanoparticles, the procedure for incubation of MNC with nanoparticles is carried out in the thermostat (37 °C) on a horizontal shaker, which provides continuous mixing of the medium.
Cells of test-cultures, without nanoparticles incubated in the same conditions, were used for determination of the initial values of the researched parameters. After incubation, samples for analysis of blood indexes are prepared (see below).
Known methods for cytological researches and tests for measurement of blood indexes are used for implementation of the invention
1. Method of flowcytometry.
Measurement of indicators selected for evaluation of the effect of nanoparticles on the state of cellular organelles, is carried out by flow cytometry.
The method of flow cytometry is based on passing the cell suspension along a capillary, through the zone of sensitivity of the device; the speed of passage of cells through the capillary is about 1000 cells/sec. In the area of the device there is no more than one cell; the cells alternately cross the focused beam of light used to excite fluorescence. The absorption and light scattering (direct light scattering with angle 0,5-2° and angular (side) light scattering at the angle of 90°) cells as well as fluorescence of markers (dyes) associated with the cell are registered with the help of light-sensitive sensors (photodiodes and photomultipliers).
The flow cytometer Beckman Coulter Epics XL (USA) is used.
The obtained information is represented with frequency histograms, which are shown in the figures.
2. Evaluation of cytotoxicity owing to viability of cells, by the method of MTT-test and the method of staining with propidium iodide (PI).
A number of living cells (under effects of nanoparticles) is estimated in the tests.
The principle of the MTT-test [Protocol of company Invitrogen (Molecular Probes), USA] is based on the ability of the enzyme of succinate d'ehydrogenase of the mitochondrial membrane of mammalian cells to restore the yellow salt 3-[4,5- dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT ) to formazan purple crystals, which are accumulated in the cytoplasm of living cells as a result of this reaction. The level of mitochondrial respiration of cells, which is an indicator of its viability, is determined according to the intensity of accumulation of crystals of formazan in the cytoplasm. The amount of formazan formed in the cell monolayer is proportional to the current amount of living cells. Formazan is extracted from cells with aprotic solvent DMSO - dimethylsulf oxide ((CH3)2SO). The intensity of the color of the solution is proportional to the amount of a live cells and is determined a colorimetric method.
A working solution of MTT (1,5 mg/ml 3-[4,5-dimethylthiazole-2-yl]-2,5- diphenyltetrazolium bromide) on the growth medium is prepared immediately before use. Cells are incubated within 2 hours in a working solution of MTT in standard conditions (+37° C, 5% C02). The reaction is stopped by removing the solution; then DMSO is added and the cells are placed on a shaker at 150 rpm for 15 minutes. The color intensity is measured on the spectrophotometer at λ = 540 nm.
MTT-test method is designed to research the adhesion of cell cultures, so this method is used in the present invention for research of multipotent mesenchymal stromal cells (MMSC). Primary culture of mononuclear blood cells (MNC), i.e. lymphocytes, is a suspension of cells, which complicates the evaluation of cytotoxicity of nanoparticles using the standard MTT-test. Regarding to it, cytotoxicity is evaluated by detecting living MNC using labeling (staining) of cells with propidium iodide (PI). The results of staining MNC are estimated on the flow cytometer Beckman Coulter Epics XL (USA).
3. Detection of disorders of the mitochondria of cells.
Disorders of the mitochondria of cells [according to Protocol of company Invitrogen (Molecular Probes), USA] were revealed owing to change of mitochondrial transmembrane potential (ΔΨιη) of tested cells, using a fluorescent potential-dependent marker (dye) MitoTracker Red 580 ( excitation = 58 1 nm, emiSsion = 644 nm), with determination of a number of stained cells and average fluorescence intensity, on the flow cytometer. MitoTracker in the final concentration of 0,5 mM was added to the tested cells of test-cultures, containing the suspension with tested nanoparticles, and to the control samples of tested cells of test-cultures containing no nanoparticles. The samples were incubated within 1 hour in standard conditions (37° C, 5% C02), then washed three times with FBS and separated from plastic with a solution of trypsin-EDTA; the suspension was centrifuged for 10 min., at 500 g; deposit was resuspended in FBS and analyzed on the flow cytometer Beckman Coulter Epics XL (USA).
4. Detection of the production of reactive oxygen species (ROS) in cells.
According to the Protocol of Invitrogen (Molecular Probes), USA, a fluorescent marker - dichlorodihydrofluorescein diacetate (H2DCF-DA) ( 10 mcg/ml); with determination of a number of stained cells and fluorescence intensity on the flow cytometer, was used for detection of the production of ROS in cells when exposed to nanoparticles. H2DCF-DA is a colorless ether which is transformed into a fluorescent product - H2DCF- when interacts with ROS. This product was visualized and the number of ROS in cells was estimated according to the intensity of its fluorescence. H2DCF-DA was added into the cell cultivated medium for 30 minutes, before determining the level of ROS in researches of intracellular level of ROS; then cells were washed twice with a medium with serum.
Mononuclear cells of blood (MNC) -lymphocytes were resuspended in phosphate buffer for the analysis on the flow cytometer. Multipotent mesenchymal stromal cells (MMSC) were separated from plastic with a solution of trypsin-EDTA. Suspensions were centrifuged within 10 min., at acceleration 500 g; the deposit was resuspended in phosphate buffer.
The production of reactive oxygen species (ROS) in cells, after effects of nanoparticles, was determined by the method of flow cytometry.
5. Determination of the state of lysosomal compartment of cells.
The principle of compartmentalization of cells suggests that biochemical processes in cells are localized in certain compartments. Most of the organelles in cells are the compartments including lysosomes.
Detection of lysosomes in cells (under the effect of nanoparticles) and evaluation of the intensity of fluorescence (which indicates activity of lysosomes) was carried out using a fluorescent pH-dependent marker (dye) LysoTracker Green DND-26 ( excitation = 504 nm, λεπύδδίοη = 511 nm), in the working concentration of 50 nM according to the Protocol of Invitrogen (Molecular Probes), USA. Cells were incubated with LysoTracker within 1 hour in standard conditions (37°C, 5% C02) and analyzed on the flow cytometer Beckman Coulter Epics XL (USA).
6. Determination of the ratio of apoptotic and necrotic cells.
A set of markers (dyes) Annexin V (Annexin V-FITC, propidium iodide (PI), binding buffer) is used for evaluation of effects of nanoparticles on the cells. It allows detection both of apoptotic and necrotic cells, according to the Protocol of Immunotech, France. In the presence of Ca2+and Mg2+, Annexin V interacts with phosphatidylserine which is shifted from the inner cell membrane to the outside one in the early stages of apoptosis. The cell membrane of living cells is impermeable to propidium iodide (PI). However, for irreversible cell damage (necrosis), propidium iodide passes through the cell membrane and interacts with the minor groove of deoxyribonucleic acid(DNA). Double staining with Annexin V and propidium iodide indicates late stages of apoptosis.
A working solution - 84 ml of distilled water, 10 ml of cold lOx buffer, 5 ml of propidium iodide, 1 ml of Annexin V per 100 ml of solution, were prepared immediately before using. Cells were washed with cold phosphate-buffered saline (PBS) and incubated in a solution within 15 min. at +4° C or on ice, without access of light. Then the cells were washed with growth medium and analyzed in the fluorescent phase-contrast microscope Leica DM5000B (filter BP 450-490, LP 520 for Annexin V; filter BP 510-560, LP 590 filter for propidium iodide). For analysis on the flow cytometer, the cells are separated from the plastic with a solution of 0,05% trypsin - 0,04% EDTA, and washed three times with PBS; then cell suspensions are stained. The results of staining are estimated by the flow cytometer Beckman Coulter Epics XL (USA). The amount of live cells, necrotic and apoptotic cells (ratio of necrotic and apoptotic ways of cell death) after the effects of nanoparticles were determined by flow cytometry.
7. Evaluation of intracellular accumulation of nanoparticles by detecting cells with changed light scattering.
Intracellular accumulation of nanoparticles is detected by the appearance of cells with a modified side light scatter, using the flow cytometer Beckman Coulter Epics XL (USA), highly sensitive sensors of the cytometer located near the flow cell, fix the light scattering angle from 2 up to 19° (it is called direct or small-angle light scattering (FSC)) and the angle of 90°, i.e. side light scattering (SSC). SSC (side scatter) is an indicator of lateral light scattering which reflects the optical heterogeneity of the cytoplasm of cells, the nature of cell inclusions and "granularity" of the cell.
For realization of the invention, tests in vitro were carried out for determination of effects of tested nanoparticles on cells of test-cultures.
In the measurement of researched blood indexes (see above mentioned items 1-7) samples of primary cultures of multipotent mesenchymal stromal cells (MMSC) and mononuclear cells - cells (MNCs), with suspensions of tested nanoparticles, according to following examples, were used:
Measurements carried out on samples, according to Examples 1-3, for MMSC test- cultures.
Example 1. In culture flasks (5 ml of growth medium in each) there are MMSC test-cultures and suspensions of nanoparticles of Si02, at concentration of nanoparticles in suspensions of 1-10"2; 1-10"3; 1·10"4% b.w..
Example 2. In culture flasks (5 ml of growth medium in each) there are MMSC test-cultures and suspensions of nanoparticles of bentonite intercalated by ions of cerium (Ce3+), at concentration of nanoparticles in suspensions of 1-10"2; 1-10"3; 1·10"4% b.w..
Example 3. In culture flasks (5 ml of growth medium in each) there are MMSC test-cultures and suspensions of nanoparticles of bentonite intercalated by ions of silver(Ag+), at concentration of nanoparticles in suspensions of 1-10"2; 1-10"3; 1·10"4% b.w.. Measurements were carried out on samples according to Examples 4-6 for MNC test-cultures.
Example 4. In culture flasks (5 ml of growth medium in each) there are MNC test- culture and suspensions of nanoparticles of Si02 at concentration of nanoparticles insuspensions: MO"2; MO"3; l-10~4b.w. .
Example 5.1n culture flasks (5 ml of growth medium in each) there are MNC test- cultures and suspensions of nanoparticles of bentonite intercalated by ions of cerium (Ce3+), at concentration of nanoparticles in suspensions of 1-10"2; 1-10"3; l-10~4 b.w. %.
Example 6. In culture flasks (5 ml of growth medium in each) there are MNC test- cultures and suspensions of nanoparticles of bentonite intercalated by ions of silver (Ag+), at concentration of nanoparticles in suspensions of 1-10 -"2 ; 1-10 -"3 ; 1-10 -4 b.w. .
Control samples of primary cultures of multipotent mesenchymal stromal cells (MMSC) and mononuclear cells (MNC) -lymphocytes, according of Examples 7 and 8 are used for carrying out researches.
Example 7. In the culture flask there is MMSC test-culture (without nanoparticles), growth medium - 4,95 ml and deionized water - 50 ml.
Example 8. In the culture flask there is MNC test-culture (without nanoparticles), growth medium - 4,95 ml and deionized water - 50 ml.
The following tests in vitro with indicators (see points 1-7) were carried out for determination of effects of tested nanoparticles on primary multipotent mesenchymal stromal cells (MMSC), separated from stromal vesicular fraction of human adipose tissue, and mononuclear cells (MNC) - lymphocytes, separated from peripheral blood of a human being in accordance with Examples 1-8:
cell viability (cytotoxicity); dysfunction of mitochondria of cells; production of reactive oxygen species (ROS); determination of the state of lysosomal compartment of cells; determination of the ratio of necrotic and apoptotic cells; intracellular accumulation of nanoparticles using fluorescent markers and flow cytometry for these purposes.
Instruments and equipment, materials and substances, methods and tests, the tested nanoparticles and cells of test-cultures and methods for their preparation used for realization of tests in vitro are described above. The results of measurements of the mentioned above indicators are submitted in Fig. 1-8 and Tables 1-4 (see above mentioned points 1-7). The results of a representative experiment for each of the tested samples (n = 3) according to Examples 1-8 are shown.
The results of researches for evaluation of viability of MMSC test-cultures are shown in Fig. 1.
The effect of tested nanoparticles on viability of MMSC was determined according to point 2.
It was revealed by the researches that all the tested nanoparticles, according to Examples 1-3, at a concentration of nanoparticles of 1-10" % b.w. provide cytotoxicity; thus for nanoparticles according to Example 1, decrease in cell viability (number of living cells%) was about 25%, whereas for the nanoparticles according to Examples 2 and 3, decrease in viability was almost 75%.
Researches of nanoparticles according to Examples 1-3, at the concentration of tested nanoparticles of 1- 10"3 % b.w. andl- 10"4 % b.w. show that the decrease in the concentration of nano particles increases (conservation) cell viability to near baseline level, corresponding to the viability of the control sample (Example 7).
The results of researches for evaluation of disorders of mitochondrial functions of MMSC are shown in Fig. 2.
Effects of tested nanoparticles on functions of mitochondria of MMSC were determined according to point 3.
It was revealed by the researches that the nanoparticles according to Example 1 did not affect the amount of MMSC in which mitochondria (cells stained with MitoTracker, %) were detected. However, at the concentration of nanoparticles of 1-10" % b.w. in Example 1, there was a significant decrease in the average intensity of intracellular fluorescence of the marker (MitoTracker/cells, conv. units). That indicates a decrease in mitochondrial transmembrane potential (ΔΨιη) of tested cells.
It was revealed by the researches that the nanoparticles according to Example 2 and 3 at the concentration of nanoparticles of 1-10" % b.w. caused decrease in the share of MMSC with detected mitochondria (cells stained with MitoTracker, % b.w.) and their transmembrane potential (ΔΨιη) decreased in dose-dependent mode, while increasing concentration of tested nanoparticles. The results of researches for evaluation of the production of reactive oxygen species (ROS) in MMSC are shown in Fig. 3.
The effect of tested nanoparticles on the production of reactive oxygen species (ROS) was determined according to point 4.
It was revealed by the researches that, according to Example 1-3, the share of MMSC (cells stained with H2DCF-DA, %) with detected reactive oxygen species (ROS) was small and did not exceed 3%. The share of MMSC (cells stained with H2DCF-DA, %) according to Examples 2 and 3 with reactive oxygen species (ROS), dose-dependent, decreases with decrease in concentration of nanoparticles.
According to Example 1 the share of MMSC (cells stained with H2DCF-DA, %) (in which reactive oxygen species (ROS) were detected) was greater at the concentration of nanoparticles of 1-10" % b.w. (in comparison with Examples 2 and 3).
Considerable changes of the average intensity of intracellular fluorescence of the marker (H2DCF-D A/cells, conv. units) were not detected in MMSC.
The results of researches for evaluation of the state of lysosomal compartment of MMSC are shown in Fig. 4.
The effect of tested nanoparticles on the state of lysosomal compartment of MMSC was determined according to point 5.
It was revealed during researches that, according to Examples 1, 2 and 3a, significant increase in the number of MMSC, in which lysosome (cells stained with LysoTracker, ) were detected, was observed at the concentration of nanoparticles of 1- 10"
2 % b.w.; this effect was less evident at the concentration of nanoparticles of 1-10 -"3 % b.w.. A number of detected dose-dependent lysosomes decreased, with decrease in the concentration of nano particles.
The average intensity of intracellular fluorescence of the lysosomal marker (LysoTracker/cells, conv. units) did not change, or slightly varied, with respect to values in the control (Example 7) at all researched concentrations of nanoparticles.
The results of researches for evaluation of the ratio of apoptotic and necrotic MMSC are submitted in Table 1. Table 1.
Evaluation of the ratio of apoptotic and necrotic MMSC after effects of nanoparticles.
Figure imgf000023_0001
Effect of tested nanoparticles on the ratio of apoptotic and necrotic MMSC were determined according to point 6.
It was revealed during researches that the tested nanoparticles according to
Example lat concentration 1- 10 -"2 ; 1-10 -"3 ; 1- 10 -~4 b.w. had no significant effect on living cells (living cells, %). The ratio of necrosis/apoptosis (Example 1) was in the range from 3,2 up to 4,4 and in the control Example 7 (without nanoparticles) the ratio of necrosis/apoptosis was 4,3. It was revealed during researches that the tested nanoparticles according to Example 2 and 3 at the concentration 1- 10" b.w.% induced considerable decrease in the share of living cells (%).
It is necessary to note that the basic way of cell death was necrosis both with (examples 1-3) or without (example 7) effects of nanoparticles.
The ratio of necrosis/apoptosis was increased considerably after exposure of nanoparticles to cells according to Examples 2 and 3 at the concentration of nanoparticles
1-10 -"2 % b.w. (Example 3) and at the concentration of nanoparticles 1-10 -"3 % b.w. (Examples 2 and 3).
The results of researches for evaluation of intracellular accumulation of nanoparticles owing to change in light scattering of MMSC are submitted in Table 2.
Table 2.
Evaluation of intracellular accumulation of nanoparticles owing to change in light scattering MMSC.
Figure imgf000024_0001
Effect of tested nanoparticles on intracellular accumulation of nanoparticles owing to change in light scattering of MMSC, was determined according to point 7.
Intracellular accumulation of nanoparticles is diagnosed owing to appearance of cells with changed (increased) light scattering.
It was revealed by the search that the tested nanoparticles according to Example 1, at concentration of 1- 10 -"2 ; 1- 10 -"3 ;1-10 -~4 % b.w., did not cause increase of light scattering of cells. Amount of cells with intact light scattering was in the range 99,5 -99,9 %; for the control example 7 (without nanoparticles) the value was 99,8%.
It was revealed during the search that tested nanoparticles, according to Examples 2 and 3, at the concentration 1-10" , show a share of cells with changed light scattering of less than 15% of the tested cells.
The results of the tests for evaluation of viability of MNC (lymphocytes) are shown in Fig. 5
It was revealed by the search that the tested nanoparticles according to Example 4, at concentration of 1- 10 -"2 ; 1- 10 -"3 ;1-10 -~4 % b.w., had no effect on cell viability.
It was revealed by the search that the tested nanoparticles according to Example 6, at the concentration of 1- 10" provided cytotoxic effect (the number of viable cells was about 75%).
It was revealed by the search that the tested nanoparticles according to Example 5, at the concentration of l- 10~2provided the death of¾ of the total population of cells.
The results of the search for evaluation of disorders of mitochondrial function of MNC (lymphocytes) are shown in Fig. 6.
Effect of tested nanoparticles on functions of the mitochondria of MNC were determined according to point 3.
It was revealed by the search that cell mitochondria (cells stained with MitoTracker, %) were detected in 100 % of living cells, not depending on the type
(Examples 4, 5 and 6) and concentration of nanoparticles (1-10 -"2 ; 1-10 -"3 ;1- 10 -~4 % b.w..) tested.
The average intensity of intracellular fluorescence of the marker (MitoTracker/cells, conv. units), indicating a change (decrease) of the transmembrane potential of mitochondria(AvPm), varied considerably depending on the type and concentration of the nanoparticles (Examples 4, 5 and 6).
It was revealed by the search that the tested nanoparticles according to Example 4, at concentration 1- 10 -"2 ; 1- 10 -"3 ;1-10 -~4 % b.w., did not provide considerable effect on the transmembrane potential of mitochondria.
It was revealed by the search that the tested nanoparticles according to Example 5, at concentration 1- 10 -"2 ; 1- 10 -"3 ;1-10 -~4 % b.w. caused a dose-dependent decrease in mitochondrial transmembrane potential. The average intensity of intracellular fluorescence of the marker (MitoTracker/cells, conv. units) according to Example 5 at the concentration of nanoparticles 1- 10" % b.w. was about 18 conventional units; in the control, without nanoparticles, (Example 8), it was about 75 conv. units.
It was revealed by the search that the tested nanoparticles according to Example 6, at the concentration 1-10" % b.w., caused decrease in transmembrane potential of mitochondria; low concentrations had less effect, but the original (control) level of transmembrane potential of cells was not obtained.
The results of search for evaluation of the production of reactive oxygen species (ROS) in MNC (lymphocytes) are shown in Fig. 7.
The effect of tested nanoparticles on the production of reactive oxygen species in MNC, was determined according to point 4.
It was revealed by the search that, according to Example 4, increase in the amount of reactive oxygen species (ROS) (intensity of fluorescence of the marker H2DCF- DA/cells, conv. units) was detected in cells at all concentrations of tested nanoparticles.
It was revealed by the search that according to Example 5, nanoparticles did not provide any effect on amount of ROS in cells.
It was revealed by the search that, according to Example 6, decrease of ROS in cells (in comparison with control) was detected at the concentration of nanoparticles of 1-10" % b.w.. At low concentrations, the level of ROS roughly corresponded to the level of ROS in control (without nanoparticles) (Example 8).
The results of the search for evaluation of the status of lysosomal compartment of MNC (lymphocytes) are shown in Fig. 8.
Effect of tested nanoparticles on the state of lysosomal compartment of MNC were determined in accordance with point 5. It was revealed by the search, that cell lysosomes (cells stained with LysoTracker, %) were detected in 100% of living cells, without depending on the type of tested nanoparticles (Examples 4, 5 and 6) and on their concentration: 1- 10 -"2 ; 1■ 10 -"3 ;1 · 10 -~4 % b.w..
The average intensity of intracellular fluorescence of the lysosomal marker (LysoTracker/cells, conv. units), which indicates activity of lysosomes, varied insignificantly with respect to the values in the control (Example 8), at all concentrations of nanoparticles tested. However, there is increased activity of lysosomes in Example 4 and a decrease in their activity according to Example 6, at concentration of nanoparticles l-10"2% b.w..
The results of the tests of evaluation of the ratio of apoptotic and necrotic MNCs (lymphocytes) are submitted in Table 3.
Table 3.
Evaluation of the ratio of apoptotic and necrotic MNC (lymphocytes) for effect of nanoparticles.
Figure imgf000028_0001
Effect of the tested nanoparticles on the ratio of apoptotic and necrotic MNCs was determined in accordance with point 6.
It is necessary to note, that the main way of cell death was apoptosis, both without effect of nanoparticles (Example 8) and with effect of nanoparticles (Examples 4-6); control cells (without nanoparticles) (Example 8) also showed a high level of apoptotic death.
It was revealed during the searches, that nanoparticles according to Example 4 did not provide considerable effect on the share of apoptotic cells, which was in the range 15,1 - 16,5 %. Nanoparticles according to Examples 5 and 6, at the concentration 1- 10" % b.w. additionally induced apoptosis in cells (respectively - 32,6 % and 31,1 ). It is necessary to note that nanoparticles according to Example 5, at the concentration 1- 10 -"2 % b.w. and 1- 10 -"3 % b.w., induced considerable necrotic death of cells (respectively - 20,7 % and 2,3%) with an overall low level of necrotic cell death.
The results of the tests for evaluation of intracellular accumulation of nanoparticles owing to change in light scattering of MNC (lymphocytes) are submitted in Table 4.
Table 4.
Evaluation of intracellular accumulation of nanoparticles owing to change in light scattering MNC (lymphocytes)
Figure imgf000029_0001
Effect of tested nanoparticles on intracellular accumulation of nanoparticles was determined, owing to change in light scattering of MNC, in accordance with point 7.
Intracellular accumulation of nanoparticles is revealed by the appearance of cells with changed (increased) light scattering. It was revealed by the search that tested nanoparticles according to Example 4, at concentration 1- 10 -"2 ; 1-10 -"3 ;1-10 -~4 % b.w. do not cause a considerable increase in light scattering of cells. Amount of cells with intact light scattering was in the range:88,8 - 90,2%; for the control example 8 (without nanoparticles) - 89,1%.
It was revealed by the search that tested nanoparticles, according to Example 5 and 6, at the concentration 1-10" % b.w., provide considerable decrease in cells with intact light scattering (respectively 39,2% and 59,9%); at lower concentrations of nanoparticles (1-10" 3and 1·10~4% b.w.) the part of cells with changed (increased) light scattering was decreased, returning nearly to the level of the control (82,1- 83,7 %).
Thus, the method of evaluation in vitro of biological effects of tested nanoparticles on cells of test-cultures on the most important indicators of cell activity was developed.
Primary cultures of multipotent mesenchymal stromal cells and mononuclear blood cells (lymphocytes) are used as cells of test-cultures. They are principal cellular elements determining the physiological tissue regeneration and repair, immune response against external stress. These important cell populations allow simulating elements of the cell system of a human being and comprehensively assessing potential biomedical risk of exposure to nanoparticles at the level of the whole organism.
The tested nanoparticles used for realization of the invention: nanoparticles of silicon dioxide (Si02), nanoparticles of bentonite (montmorillonite) intercalated by ions of silver (Ag+) and nanoparticles of bentonite (montmorillonite) intercalated by ions of cerium (Ce3+) are perspective nanomaterials for medical, pharmacological, cosmetic ant technical purposes. Tested nanoparticles allow evaluating various levels of their biological effect on cells of test-cultures, due to selected indicators of cell activity.
The methods developed for evaluation in vitro of biological effects of tested nanoparticles on cells of test-cultures, allow reliably estimation of the effect of the type of nanoparticles and their concentration (concentration effect) on indicators of cell activity and allow to take a decision about the possible risks of using biomedical nanoparticles.

Claims

1. Method to evaluate in vitro the biological effect of bactericidal inorganic nanoparticles on the cellular system of a living organism, essentially consisting in the determination of a selected set of indexes on primary cell cultures of multipotent mesenchymal stromal cells separated from stromal-vascular fraction of human adipose tissue, and on mononuclear cells-lymphocytes separated from human peripheral blood, in the presence of suspensions containing various amounts of the tested nanoparticles, then compairing the obtained values of the indexes with the values of the same indexes determined in control samples of the same cell cultures, without the presence of the suspension of nanoparticles, and assessing comprehensively the figures obtained.
2. Method according to claim 1 where the set of biological indexes to be determined, comprises:
cell viability (cytotoxicity)
dysfunction of mitochondria of cells
production of reactive oxygen species (ROS)
state of lysosomal compartment of cells
ratio of necrotic and apoptotic cells
intracellular accumulation of nanoparticles
3. Method according to claim 2 where the set of biological indexes is determined using fluorescent markers (dyes) and flow cytometry.
4. Method according to claim 1 where the indexes are determined by incubation of the cell test-cultures with the suspensions of the tested inorganic nanoparticles, within 12-36 hours.
5. Method according to claim 4 where 1% aqueous suspensions of nanoparticles are admixed with the cells growth medium in a ratio 1: 100, 1: 1000, 1: 10000 so as to have a concentration of nanoparticles of 1.10 -"2 , 1.10 -"3 , 1.10 -"4.
6. Method according to claim 4 where inorganic nanoparticles are used selected in the group comprising silicon dioxide (Si02) with a size of the particles of 5-12 nm, bentonite (montmorillonite) nanoparticles intercalated with silver ions (Ag+) with a size of nanoparticles of 20-200 nm, bentonite (montmorillonite) nanoparticles intercalated with cerium ions (Ce3+) with a size of the particles of 20-200 nm.
7. Method according to claim 2 where the index of cytotoxicity of the tested nanoparticles is determined by submitting their suspension in a growth medium of mesenchymal, stromal cells to the MTT-Test, using a solution of 3-[4,5- dimethylthiazole-2-yl] -2,5-diphenyltetrazolium bromide.
8. Method according to claim 2 where the index of cytotoxicity of the tested nanoparticles is determined by submitting their suspension in a growth medium of mononuclearcells-lymphocytes to determination of necrotic cells by staining with the marker propidium iodide(PY) and then to analysis on the flow cytometer.
9. Method according to claim 2 where the index of disfunction of mitochondria of cells is determined measuring the change of mitochondrial transmembrane potential of tested cells, using the potential-dependent marker Mito Tracker Red 580, with determination of the number of stained cells and of average fluorescence intensity on the flow cytometer.
10. Method according to claim 2 where the index of production of reactive oxygen species (ROS) in cells of test-cultures, is determined by using the fluorescent marker dichlorodihidrofluorescein diacetate (H2DCF-DA) with determination of the number of steined cells and of fluorescence intensity on the flow cytometer.
11. Method according to claim 2 where the index of the state of lysosomal compartment of cells is determined by detecting lysosomes in cells and by evaluating intensity of fluorescence by using a fluorescent pH-dependent marker Lyso Tracker Green DND-26 and the flow cytometer.
12. Method according to claim 2 where the index of the ratio of necrotic and apoptotic cells is determined using the set of markers of Annexin V, with analysis on the floweytometer to detect both apoptotic and necrotic cells.
13. Method according to claim 2 where the index of intracellular accumulation of nanoparticles is determined by measurement of side light scattering using the floweytometer.
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