Nanoceria-curcumin conjugate: Synthesis and selective cytotoxicity against cancer cells under oxidative stress conditions.

A water- and alcohol-soluble cerium oxide-curcumin conjugate was obtained by co-evaporation with poly(N-vinylpyrrolidone) (PVP). A nanocomposite consisting of hybrid organic-inorganic particles was stable in a wide range of pH values. Its properties were evaluated using nine cell lines: normal (MDBK, ST, Vero) and malignant (L929, T98G, HEp-2, A549, RIN-m5F, Hep G2). PVP-stabilised nanoceria was shown to inhibit autoxidation of curcumin, to enhance curcumin photostability, to promote bioaccumulation and to affect curcumin cytotoxicity and photocytotoxicity, depending on cell type, being more toxic to cancer cells in a selective manner. Under the conditions of UVA/UVC or H2O2-induced oxidative stress, the nanoceria-PVP-curcumin (NPC) conjugate was found to possess a selective cytotoxicity: it caused drastic inhibition of metabolic activity or a decrease in the total number of tumour cells, while in non-transformed cultures under the same conditions, the nanoceria-PVP-curcumin conjugate protected cells from these damaging factors. The NPC-conjugate, unlike curcumin itself, demonstrated a photosensitising effect in tumour cell cultures, while protecting non-transformed cultures from the damaging effects of UV radiation or oxidative stress. Based on the results obtained, we strongly believe that this novel hybrid material has enhanced characteristics compared to other curcumin formulations, and can be considered as a potent drug for biomedical applications, including cancer therapy.

Photo-induced toxicity of tungsten oxide photochromic nanoparticles.

We synthesised a new type of photochromic tungsten oxide nanoparticles, analysed their photocatalytic activity and carried out a thorough analysis of their effect on prokaryotic and eukaryotic organisms. Ultrasmall hydrated tungsten oxide nanoparticles were prepared by means of hydrothermal treatment of tungstic acid in the presence of polyvinylpyrrolidone as a template, stabiliser and growth regulator. Tungstic acid was synthesised through an ion-exchange method using sodium tungstate solution and a strongly acidic cation exchange resin. Upon illumination, photochromic nanoparticles of WO3 were shown to increase greatly their toxicity against both bacterial (both gram-positive and gram-negative - P. aeruginosa, E. coli and S. aureus) and mammalian cells (primary mouse embryonic fibroblasts); under the same conditions, fungi (C. albicans) were less sensitive to the action of tungsten oxide nanoparticles. UV irradiation of primary mouse fibroblasts in the presence of WO3 nanoparticles demonstrated a time- and dose-dependent toxic effect, the latter leading to a significant decrease in dehydrogenase activity and an increase in the number of dead cells. WO3 nanoparticles were photocatalytically active under both UV light and even diffused daylight filtered through a window glass, leading to indigo carmine organic dye discolouration. The obtained experimental data not only show good prospects for biomedical applications of tungsten trioxide, but also demonstrate the need for clear control of biosafety when it is used in various household materials and appliances.

OBTAINING OF THE TRANSGENIC HELIANTHUS TUBEROSUS L. PLANTS, CALLUS AND "HAIRY" ROOT CULTURES ABLE TO EXPRESS THE RECOMBINANT HUMAN INTERFERON ALPHA-2b GENE.

This work is the first to our knowledge to describe the successful attempt of Agrobacterium rhizogenes-mediated transformation of topinambour in order to obtain the transgenic H. tuberosus plants, callus and "hairy" root cultures. The plasmid vectors contained the sequence of interferon gene fused with Nicotiana plumbagenifolia L. calreticulin apoplast targeting signal driven by 35S CaMV promoter or root-specific Mll promoter. Nearly 75% isolated Ri-root lines and callus cultures were proved (by PCR analysis) to contain HuINFa-2b transgene. We also managed to obtain H. tuberosus transgenic plants through somatic embryogenesis on the transgenic "hairy" root culture. The obtained transgenic H. tuberosus cultures exhibited high-level antiviral activity that ranged from 2000 to 54500 IU/g FW that makes this crop considered a promising source of recombinant interferon alpha 2b protein.

[THE INFLUENCE OF NANODISPERSE CERIUM DIOXIDE ON ONTOGENETIC CHANGES OF ANTIOXIDANT SYSTEM IN THE MUCOSA OF THE STOMACH AND COLON IN RATS].

It was established that with age the content of lipid peroxidation products increased in the mucosa of the stomach: Diene conjugates by 30%, products which react to thiobarbituric acid by 285% and Schif bases by 181%. Nanodisperse cerium dioxide (NCD) reduced the content of lipid peroxidation in the gastric mucosa in old rats: Diene conjugates by 43 %, products which react to thiobarbituric acid by 51% and Schif bases by 44% relative to the control group of rats given age. Similarly, it was established that the content of Diene conjugates increased by 40%, products which react to thiobarbituric acid by 114% and Schif bases by 132% in the mucosa of the colon of old rats. NDC significant reduced the content of products which react to thiobarbituric acid by 69% and Schyf basics by 132%. In the stomach superoxide dismutase (by 43%) and catalase activity (by 24%) decreases with age, while in the colon superoxide dismutase activity increases (by 43%). In the colon NCD significant decreased superoxide dismutase (by 34%) and catalase activity (by 21%) relative to controls. Thus, the NDC restores lipid peroxidation in the gastric mucosa and colon, in which develops oxidative stress with age.

[The changes of the motor function of the stomach and the colon under the action of the nanocrystalline cerium dioxide].

We investigated the effect of nanocrystalline cerium dioxide on parameters of spontaneous and stimulated motility of the stomach and colon in rats. It was found that administration of nanocrystalline cerium dioxide for 10 days increased the amplitude of contractions of stimulated motility in the stomach by 33.0 +/- 2.4% and the frequency of contractions of the colon by 80.3 +/- 7.5%. In this group, the introduction of carbachol also increased the frequency of the contractions by 274.0 +/- 22.9%, compared to the control group. The administration of nanocrystalline cerium dioxide increased the index of motor activity of spontaneous and stimulated motility of the stomach by 19.8 +/- 1.4 and 14.5 +/- 9.0%, respectively. In the colon, the motor activity increased by 14.3 +/- 1.1 and 11.1 +/- 0.8%, respectively. We also found that the nanocrystalline cerium dioxide rebuilt morphological condition of the mucous of the colon.

[Morphofunctional state of reproductive system of ageing male rats in case of using nanocerium].

The influence of nanocrystalline cerium dioxide (NCD, 1 and 100 mg/kg per os daily for 10 days) on morphofuctional state of reproductive system was investigated in ageing male rats. It has been established that activation of hormone-producing testicular Leydig's cells, as well as of secretory and proliferative processes in prostate, underlies the stimulating effect of NCD at a dose 1 mg/kg on hormonal function of testis and spermatogenesis of ageing male rats. NCD used at a dose 100 mg/kg had no significant effect on the assessed indices of morphofuctional state of reproductive system.

Panthenol-stabilized cerium dioxide nanoparticles for cosmeceutic formulations against ROS-induced and UV-induced damage.

A method of panthenol-stabilized cerium dioxide nanoparticles synthesis was developed and their effect on the survival rate of human epidermoid cancer cells HEp-2 and diploid epithelial swine testicular cell line (ST-cells) under oxidative stress conditions induced by hydrogen peroxide introduction and UV irradiation was studied. The results obtained indicate that the use of panthenol as a stabilizer supposedly provides a substantial increase in the efficiency of protection. The degree of protection is determined by panthenol-to-ceria molar ratio. The combination of panthenol and nano-ceria protects biological objects under study from reactive oxygen species (ROS) and UV-irradiation more effectively than individual panthenol or ceria. The protective action of panthenol-stabilized cerium dioxide nanoparticles depends strongly on their composition and the means of their application.

[Effects of diphenyl derivatives on electrophoretic mobility of murine T lymphocytes].

The early changes of electrophoretic mobility (EPM) of murine T lymphocytes induced by structural analogues of amixine-dihydrochloryde 4,4'-bis-[2(diethylamino)ethoxy]diphenyl (compound 1) and dihydrochloryde 2-methoxycarbonil-4,4'-bis-[2(diethylamino)ethoxy]diphenyl (compound 2) were studied by electrophoresis technique. During the interval 0-2 hours all compounds increased the absolute values of EPM in comparison with control. These changes were of the same kind--distinctions were quantitative. Amixine and compound 1 during the interval 2-4 hours additionally increased the EPM. The compound 2, on the contrary, decreased the EPM. It was shown that the opposite effects of the aforementioned compounds were caused by the fact that amixine and compound 1 induce, and compound 2 does not induce IFN production in T lymphocytes in vitro. The results of our experiments are important for understanding of the mechanisms of immunomodulating effect of amixine and its structural analogues.

Dose-dependent IFN-stimulating and immunomodulating properties of 6H-indolo[2,3-B] quinoxaline derivatives.

Two 6H-indoloquinoxaline derivatives were studied in different doses and schemes of application for their INFgamma-inducing potential and ability to effect functional activity of phagocytic cells. Tested compounds were shown to possess comparable or higher activity than reference drug Amixin in analogous doses. One indoloquinoxaline significantly elevated metabolic activity of macrophages and increased their potential for phagocytosis. Application of multiple treatments and higher doses allowed us to reveal differences between studied derivatives that were not obvious in previous in vivo experiment. Capacity of 6H-indoloquinoxalines to induce vast IFN amounts on in vivo level was demonstrated for the first time.

Effect of tilorone and its analogues on the change of mitochondrial potential of rat hepatocytes.

The influence of tilorone dihydrochloride and its analogues--diphenyl derivatives on the changes of transmembrane potential of mitochondrial membranes of the isolated rat hepatocytes has been estimated. Authors have shown a significant increase in mitochondrial potential thirty minutes after the introduction of the test compounds to the cells using the fluorescent probe JC-1. These results indicate the rapid activation with tilorone and its analog--dihydrochloryde 4,4'-bis-[2-(diethylamino)ethoxy]diphenyl--of the RLR signaling pathway. The final stage of this pathway is the cell production of IFN type I. The authors concluded that there is an increasing of the organelles resistance to the extra/intracellular damaging agents under the influence of the test compounds.

[Production of purified human recombinant alpha2b-interferon].

Technology for producing biologically active recombinant alpha2b-interferon is based on creating a bacterial producer strain containing the cDNA of human interferon alpha. The authors have obtained two producers of recombinant alpha2b-interferon, the synthesis of the target protein in them occurs in the inclusion bodies. The schemes of isolation and purification of biologically active recombinant alpha2b-interferon have been developed. The drug purity was approximately 97-98%. Biological activity in the culture of sensory cells in the cytopathic test was 4.2*10(8)ME/mg.

Antibacterial activity of cerium colloids against opportunistic microorganisms in vitro.

The CeO2 sol with the size of nanoparticles 2-4 nm has been synthesized. It has been determined that the synthesized nanocrystalline cerium has antibacterial activity in vitro against different groups of opportunistic microorganisms: clinical strains of Escherichia coli, Staphylococcus aureus and Candida albicans. The rate of viability depression of test-cultures depends on the concentration of cerium dioxide nanoparticles and time of incubation. It is shown that the sol interacts with the bacterial cell surface. It is suggested that the observed differences of antibacterial action of nanocrystalline cerium dioxide can be related to the structural characteristics of the cell surface.

[The antiviral activity of diphenyl derivatives in different model systems].

In experiments on two model systems L929/VSV and RF/ HSV-1 in vitro we have studied the antiviral activity of new structural analogues of tilorone--4,4'-bis[2-(diethyl-amino)ethoxy]diphenyl dihydrochloride and 2-methoxy-carbonyl-4-4'-bis[2-(diethylamino)ethoxy]diphenyl dihydrochloride. In experiments the tested substances were administered in preventive and therapeutic schemes. The preventive scheme confirms the existence of a correlation between IFN production under the influence of tested substances and their antiviral activity. It is shown that diphenyls are able to inhibit the development of viral cytopathic effect induced by DNA- and RNA-containing viruses. Diphenyl derivatives are less toxic than tilorone and could be considered as promising substances for further research to develop new antiviral drugs.

UV-shielding property, photocatalytic activity and photocytotoxicity of ceria colloid solutions.

UV-shielding property, photocatalytic activity and cytotoxicity (including photocytotoxicity) of citrate-stabilized ceria colloid solutions were studied. It was established that UV-shielding property (namely, the sun protection factor, the critical absorption wavelength and the UVA/UVB-ratio) of ceria nanoparticles are as good as those of titanium dioxide and zinc oxide nanoparticles. It was further demonstrated that ceria nanoparticles possesses substantially lower photocatalytic activity, which additionally decreases upon decrease in ceria particle size. It was found that colloid ceria solutions are non-toxic to mouse fibroblasts (L929) and fibroblast-like cells of African Green monkey (VERO). Moreover, ceria nanoparticles are capable to protect these cells from UV-irradiation-induced damage. It was proposed that nanocrystalline ceria could be used not only as UV-blocking material, but also as prophylactic and even therapeutic compound for sunburns treatment.

[Nanocrystaline ceria based materials--perspectives for biomedical application].

Nanocrystalline ceria possesses a unique complex of physical and chemical properties making it highly bioactive material. In this review, modern data on the action of nanocrystalline ceria on cells, micro- and macroorganisms are analyzed. Special attention is paid to the analysis of the factors affecting protective properties of CeO2 with respect to the living systems.

[Effect of amyxine, loramyxine and their composites with yeast RNA on spontaneous and induced mutagenesis in Salmonella typhymurium].

It was found out that amyxine, loramyxine and their composites with yeast DNA do not increase reliably the frequency of spontaneous mutations in test-strain S. typhymurium TA 98. The studied substances do not possess the antimutagen activity in respect of mutations, induced by potassium bichromate in S. typhymurium TA 98 and do not increase their quantity.

[Antiviral effect of cerium dioxide nanoparticles stabilized by low-molecular polyacrylic acid].

Antiviral activity of nanosize cerium dioxide sols (CeO2) in animal cell culture has been studied. The inhibiting effect of the mentioned sols upon reproduction of vesicular stomatitis test-virus was demonstrated for the first time in case of preliminary 24-hour contact with cells lines L929 and EPT. The effectiveness of protective action depends on initial precursor and the way of obtained water nanosize nanosols.

[Effect of alpha/beta-interferon on electrophoretic mobility of murine T-lymphocytes].

Alpha/beta-Interferon (alpha/beta-IFN)-induced early changes in the electrophoretic mobility (EPM) of murine splenic T lymphocytes were studied by the microelectrophoresis technique. It has been found that alpha/beta-IFN-treated T lymphocytes have a greater EPM than control cells within first hours of alpha/beta-IFN addition. This change in EPM depends on the concentration of alpha/beta-IFN in the medium and the duration of alpha/beta-IFN interaction with the cells. It is concluded that the alpha/beta-IFN-treated cells have a greater net negative charge of the cell surface than control cells.

[Fundamentals of interferon system function in pathology and molecular biological peculiarities of interferon production].

Molecular biological peculiarities of interferon system function in PV-infected persons have been found. It is evident that the interferon production, anti-inflammatory cytokines and their receptors and also defensines play an important role in the mechanism of virus interaction with sensitive cells of macroorganism with development of pathological process. The new conception of expediency for the use of interferons and their inducers as the polyfunctional regulators with a broad spectrum of activity for the treatment of PV-infected patients was suggested. Patents for the method of treatment of PV-infected patients were obtained. New inducers of interferon as well as recombinant IFN-alpha-2b was developed. Our results were introduced in the medical practice.

[Interferon-stimulating activity of muramoyl dipeptide glycosides].

The uninvestigated interferon (IFN)-inducing capacity of glycoside derivatives of N-acetylmuramoyl-L-alanyl-D-isoglutamine (MDP) has been studied. Most MDP glycosides tested in vitro were more active than the reference preparation MDP. Under the in vivo conditions three studied preparations: MDP, a-heptyl-MDP and beta-(2-methyl-3-phenylchromonyl-7)-MDP stimulated production of considerable amount of circulating IFN that evidences for the promising character of their further investigation as preparations immunostimulators.