Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo.

UNLABELLED: The aim of the work was the synthesis of gold nanoparticles (GNP) of different sizes and the estimation of their biological activity in vitro and in vivo. MATERIALS AND METHODS: Water dispersions of gold nanoparticles of different sizes have been synthesized by Davis method and characterized by laser-correlation spectroscopy and transmission electron microscopy methods. The GNP interaction with tumor cells has been visualized by confocal microscopy method. The enzyme activity was determined by standard biochemical methods. GNP distribution and content in organs and tissues have been determined via atomic-absorption spectrometry method; genotoxic influence has been estimated by "Comet-assay" method. RESULTS: The GNP size-dependent accumulation in cultured U937 tumor cells and their ability to modulate U937 cell membrane Na(+),K(+)-АТР-ase activity value has been revealed in vitro. Using in vivo model of Guerin carcinoma it has been shown that GNP possess high affinity to tumor cells. CONCLUSIONS: Our results indicate the perspectives of use of the synthesized GNP water dispersions for cancer diagnostics and treatment. It's necessary to take into account a size-dependent biosafety level of nanoparticles.

The prospects of medical application of metal-based nanoparticles and nanomaterials.

Current studies, dedicated to metallic (gold, silver, iron, and copper) nanomaterials are reviewed in this paper. This metals own unique physical and chemical properties which determine their application. The medical application of metallic nanomaterials includes therapy and prophylaxis of diseases, development of new drugs and improvement of conventional ones, nanodiagnostics. Nevertheless some aspects concerning the introduction of the nanometals into medical practice need further profound research.

Peculiarities of live cells' interaction with micro- and nanoparticles.

Experimental evidence collected more than 20 years ago in different laboratories suggests that the interactions between live biological cells and micro- and nanoparticles depend on their metabolic state. These experiments were conducted by reputable groups, led by prominent leaders such as H. Pohl of the USA, who was the inventor of dielectrophoresis, and B. Derjaguin of the Soviet Union who was the leading author of DLVO theory. The experiments had been mostly conducted with microparticles in the early 1980s. In the early 1990s, Ukrainian researchers showed that the interaction of live cells with gold nanoparticles consisted of an initial reversible step that also depended on cell metabolism. They found indirect evidence that the ion pumps of the cells were responsible for the reversible step. Ion pumps generate a transmembrane potential, a measurable and widely-used characteristic of the cell's energetic state. The transmembrane potential, in turn, strongly affects the zeta-potential, as was experimentally discovered 40 years ago by several independent groups using cell electrophoresis. This relationship should be taken into account when DLVO theory is considered as the basis for describing the interactions between live cells and micro- and nanoparticles. Unfortunately, detail theoretical analysis indicates that such modification would not be sufficient for explaining observed peculiarities mentioned above. That is why distinguished theoreticians such as Pohl, Frohlich, Derjaguin and others have suggested three theoretical models, presumably to explain these experiments. These theoretical models should be considered to be complementary to the well-established concepts developed on this subject in the molecular biology of cells and cell adhesion. This paper is not a revision of the existing models. It is an overview of the old and forgotten experimental data and discussion of the suggested theoretical models. The unusual interaction mechanisms are only specific for live biological cells and serve a dual role: either as a first barrier to protect the cell from potentially damaging, dispersed particulates, or as a means of accumulating useful substances. Both functions are critical for the modern problem of nanotoxicology.

[Obtaining of bacterial bioluminescent strain Protobacterium phosphoreum B7071 (lux+) for the determination of zinc ion concentration].

The transconjugate containing hybrid plasmid (Te(R)Zn(R)lux+) was obtained using the conjugation method on Pseudomonasfragi T2(5) (Te(R)ZnR) strain and bioluminescent strain Protobacterium phosphoreum B7071 (lux+). The expression regulation of lux-genes on the obtained plasmid is carried out by promotor-operational area conjointly with zinc resistance genes. The cells of the obtained genetically modified bacteria have the ability to specific induced luminescence, which is a respond to zinc ions' presence in the measuring medium. It was shown that the cells' bioluminescence intensity of trans-conjugate is linearly dependent on zinc ions' concentration within the range of 1-100 microM, that provides the opportunity of using biosensor as a strain for qualitative and quantitative detection of the metal. The low sensitivity limit of this method is 0.5 microM for the metal. Besides having high sensitivity, the developed lux-biosensor is highly specified.

[Structural and functional aspects of bacterial membranes and liposomes interactions].

Effect of exogenous lipids on the morphology and enzymatic activity of Bacillus cereus B4368 membrane has been studied. Specific character of different lipids action on the membrane enzyme activity was found. This peculiarity had been taken into account by using liposomes in biological systems.

[Effect of colloid gold on physiologic-biochemical processes in Escherichia coli 1257]. / Vliianie kolloidnogo zolota na fiziologo-biokhimicheskie protsessy Escherichia coli 1257.

The influence of colloid gold on the growth processes, ATP-ase activity and extrusion of protons in Escherichia coli 1257 was studied. The particles of colloid gold exert nonmonotonous influence on these processes with different direction is such a way that small concentration of this metal (5 x 10(-7)-5 x 10(-6) mg/ml) exert stimulative effect, while higher concentrations of colloid gold result in the suppression of biological activity of the bacterial cells. The discovered peculiarities of colloid gold influence of E. coli strain may be determined by specificity of contact interaction of metal particles with the surface of bacterial cells.

[Obtaining bioluminescent bacterial strains for detection of thallium ions]. / Poluchenie bioliuminestsentnykh shtammov bakterii dlia detektsii ionov talliia.

The possibility of Alcaligenes eutrophus T1 luxphenotype creation by temperature induced mutagenesis has been shown. These mutants are able to emit light after induction by thallium ions. This biological method of thallium detection possesses high specificity and sensitivity (0.5 microM of metal). Such microbial cells can be used to quantify bioavailability of thallium part in the environment.

[Inhibitory effect of lead ions on cells of some strains of Pseudomonas species bacteria]. / Izuchenie ingibiruiushchego vliianiia ionov svintsa na kletki nekotorykh shtammov bakterii roda Pseudomonas.

The inhibition effect of ionic lead on membrane ATPase activity, transmembrane potential (delta psi) and permeability level of the Pb-sensitive P. fluorescens B894 and Pb-resistant P. fluorescens B4252 bacteria cells have been studied. It have been shown that decreasing ATPase activity and transmembrane potential values and the increasing of permeability by lead are higher for Pb-sensitive strain then for Pb-resistant. It is suggested that mechanism of the ionic lead toxic effect deals with plasma membrane biochemical parameters (ATPase activity, value of delta psi) alterations and interruption of it barrier function.

[Effect of arsenic on bacterial growth and plasma membrane atpase activity]. / Osobennosti vliianiia mysh'iaka na rost bakterii i ATFaznuiu aktivnost' plasmaticheskikh membran.

The effects of arsenic in the forms of arsenite and arsenate on bacterial growth and plasma membranes ATPase activity of was studied. Correlation of The rate of ATP hydrolysis was found to be correlated with bacterial resistance to toxic arsenic ions. Detoxification of arsenate by resistant cultures of bacteria was suggested to be related with an increase in bacterial ATPase activity and the degree of ATPase mobilization.

[Inducible surface proteins from some strains of Pseudomonas species bacteria while adapting to cobalt]. / Indutsibel'nye poverkhnostnye belki nekotorykh shtammov bakterii roda Pseudomonas v usloviiakh adaptatsii k kobal'tu.

The possibility of increasing resistance of some Pseudomonas strains to cobalt at adaptation to monotonous increasing its concentration was studied. Strains Pseudomonas fluorescens B5242 and Pseudomonas fluorescens B894 are capable to increase its resistance in such conditions via inducible synthesis of protective surface proteins. The molecular masses of such proteins were 55.0; 45.0 and 33.0 kDa for P. fluorescens B5242 strain.

[Effect of thallium of ATP-ase activity and transmembrane potential in bacteria]. / Vliianie talliia na ATP-aznuiu aktivnost' i transmembrannyi potentsial bakterii.

Effect of thallium (TlNO3) on the ATPase activity and transmembrane potential (Dj) of bacteria with different levels of resistance to this metal has been studied. The hypothesis has been made that the resistance biochemical mechanism is based on the energy transformation systems in the cell.

[Transmembrane potential and ATP-ase activity of the plasma membrane of bacteria exposed to heavy metals]. / Transmembrannyi potentsial i ATP-aznaia aktivnost' plazmaticheskoi membrany bakterii pri vozdeistvii tiazhelykh metallov.

Effect of heavy metals (gold, zinc and cadmium) on the ATPase activity and transmembrane potential (delta psi) as integral index of bacterial cells membrane processes intensity has been studied. It was shown that studied membrane systems of bacteria are very sensitive to the action of heavy metals and they can serve as indicators of resistance of bacterial cultures.

[Bacterial cell membrane ATPase in assessing heavy metal toxicity]. / ATFaza plazmaticheskikh membran bakterii v otsenke toksichnosti tiazhelykh metallov.

Sensitivity of membrane ATPase to heavy metals was studied in bacterial stains Bacillus cereus ATCC 14579, Bacillus cereus B4368, and Alcaligenes eutrophus CH34 was studied. The inhibition effects of metals on membrane ATPase ranged in the same order (Au > Cu > Zn > Co > Mn) as their effects on the growth of cultures. It is suggested that membrane ATPase is one of the targets for the action of heavy metal ions within the microbial cell and, therefore, its activity may serve as an indicator of their toxicity.

Interaction of energized bacteria cells with particles of colloidal gold: peculiarities and kinetic model of the process.

It is found that the cells of Bacillus cereus B-4368 at energized state can concentrate the colloidal gold particles on their surface. It is shown that the process depends on metabolic reactions proceeding on the plasma membrane. The inhibitory analysis permits to suppose that the metal concentration is due to the functioning of ATP-dependent generator of the transmembrane potential, apparently, of proton ATPase. Kinetic characteristics of the process show the presence of an intermediate state in the formation of biomineral aggregates. A kinetic model of the studied process is suggested which describes the experimental data well.

[The effect of respiratory toxins on the bacterial concentration of trivalent gold]. / Vpliv dikhal'nykh otrut na kontsentruvaniia bakteriiami trivalentnogo zolota.

It has been found that concentration of trivalent gold by Bacillus cereus V-4368 cells is sensitive to respiratory inhibitors such as rotenone and antimycin A. Inhibitory action of rotenone is neutralized by succinate, that of antimycin--by ATP. ATP influence disappears in presence of DCCD. It is supposed that Au(III) concentration is due to the ATPase action and the respiration systems supply this process with ATP.

[The role of membrane processes in Au(III) and Au(0) accumulation by bacteria]. / Rol' membrannykh protsessov v nakoplenii Au(III) i Au(0) bakteriiami.

The role of structural and functional factors in the processes of the bacterial cell interaction with colloid Au (0) and ionic Au (III) states has been investigated. It is shown that the bacterial walls of Bacillus sp. 4368 aggregating with colloid gold contain glycoprotein with isoelectric point 11. Glycoprotein from cell walls indifferent to colloid gold strain (Bacillus subtilis 168) has pHiso = 5. At the same time the cells of both strains accumulate Au (III) introduced into a medium in the form of tetrachloroaurate. The process is energy-dependent because it is suppressed by azide, uncouplers of oxidative phosphorylation and dicyclohexyl carbodiimide (DCCD). The role of ATPase of Au (III) accumulation has been studied on Bacillus sp. 4368 plasma membrane vesicles. The ATPase activity is inhibited by 70, 50 and 35-50% by vanadate, DCCD and Au (III), respectively, but it does not change in the presence of dinitrophenol and NaN3. ATP but not ADP and AMP stimulated the Au (III) accumulation by membrane vesicles and prevents the inhibitory action of azide but neither of DNP or DCCD. In the energized state membrane vesicles link gold sol particles. It has been assumed that the Au (III) accumulation is associated with the functioning of transmembrane potential generators, the metal being localized on the membrane surface.

[Interaction of the glycoprotein from the Bacillus pumilis cell wall with liposomes]. / Vzaimodeistvie glikoproteida kletochnoi obolochki Bacillus pumilis s liposomami.

The methods of centrifugation and gel-filtration on Sephadexes G-50 and G-150 were used to study the interaction of Bacillus pumilis cell wall glycoprotein component having the molecular weight of 50 kDa (GP-50) with lyposomes from bacterial lipids. GP-50 is shown to sorb on such liposomes and disturb their barrier properties inducing yield of low-molecular label. GP-50 exerts no effect on properties of liposomes from egg lecithin. Electrostatic forces are supposed to play a decisive role in initial acts of GP-50 interactions with lipid phase of microbial envelopes.

[Effect of n-chloromercuribenzoate and albumin on Mg2+, Ca2+-ATPase activity of plasma membranes of smooth muscle cells]. / Vliianie n-khlormerkuribenzoata i al'bumina na Mg2+, Ca2+-ATP-aznuiu aktivnost' plazmaticheskikh membran gladkomyshechnykh kletok.

It is shown that Mg2+, Ca2+-ATPase activity of plasma membrane fragments from the rat small intestine myocytes is inhibited by p-chloromercuribenzoate and dithionitrobenzoate (50 and 90%, respectively). The effect of p-chloromercuribenzoate inhibition is removed by serum albumin promoting a rise in the ATPase activity of the plasma membranes and shifting the temperature maximum point up to 32 degrees C (in the norm the maximum is observed at 36 degrees C). According to the data presented, albumin changes the composition and phase state of the lipid surrounding of the membrane enzymes.

[Mg2,Ca2+-ATPase activity of sarcolemmas of intestinal smooth muscle cells in the rabbit]. / Mg2+,Ca2+-ATPaznaia aktivnost' sarkolemmy gladkomyshechnykh kletok tonkoi kishki krolika.

Preparations of rabbit small intestine smooth muscle cell sarcolemma are capable of hydrolyzing ATP in the presence of millimolar concentrations of Mg2+ and Ca2+ and possess the activity of Mg2+,Ca2+-ATPase having a high affinity for Ca2+ (Km = 5.8 X 10(-6) M). The optimal conditions for the Mg2+,Ca2+-ATPase reaction were established. It was demonstrated that sarcolemmal preparations hydrolyze ATP, GTP, ITP and UTP almost at the same rates. The enzyme contains SH-groups that are unequally exposed to the water phase and are inhibited by 50% by p-chloromercurybenzoate and by 90% by dithionitrobenzoate. The Mg2+,Ca2+-ATPase activity is highly sensitive to oxytocin: at the concentration of 10(-7) MU/ml, the hormone completely inhibits the enzyme without affecting its Mg2+-, Ca2+- and Na+,K+-ATPase activities.

[Extraction of plasma membranes from smooth muscle cells of the rabbit small intestine]. / Vydelenie plazmaticheskikh membran kletok gladkikh myshts tonkoi kishki krolika.

A method is offered for isolation of subcellular fractions from small intestinal smooth muscle cells enriched by plasma membranes (PM). The method is based on differential centrifugation over sucrose density gradient. According to the localization of marker enzymes, the membrane fraction obtained with the use of 30% sucrose is considered to be optimal. The PM fraction is superior to the homogenate 10-fold on the average in the magnitude of Na, K-ATPase, 17-fold in Mg2+-ATPase, and 15-fold in that of 5'-nucleotidase activity. ATPase of PM is activated by Ca2+ in micro- and millimolar concentrations. It is suggested that Mg2+-dependent Ca-activated ATPase of PM is related to the Ca2+ content control in the cell.