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Project titleDevelopment of biologically active nanomaterials for medical and veterinary purposes

Research area 03 - CHEMISTRY AND MATERIAL SCIENCES, 03-601 - Chemistry of new inorganic functional and nano-dimensional materials

Keywordsnanoparticles, silver, gold, medical preparations, veterinary preparations

Annotation
The explosive development of nanotechnology in recent years has led to the emergence of a number of unique materials used in critical areas of the economy and social sphere. One of such directions was the active development of nanomedicine - the production of fundamentally new drugs and medical devices based on nanomaterials of various types. One of the most striking examples of such materials was the synthesis of medical and veterinary drugs based on silver nanoparticles, as well as a number of other metals - Au, Pt, etc. A large number of scientific groups in developed countries are actively developing new methods for the synthesis of such nanomaterials, studying their properties and methods of application. The appearance of such nanopreparations has opened a new page in the fight against a number of socially significant diseases. Currently, there is a real and fairly high probability of new infections that pose an increased danger to humans, as well as domestic animals and agricultural plants. The precursors of these infections tend to circulate among wild animals and birds. The high contagiousness of new infectious pathogens significantly increases the risk of epidemics and global pandemics. Population growth, its crowding in big cities, constant migration, tourism, business trips - all this also increases the likelihood of rapid spread of infections. The recommended measures to limit the spread of infections – quarantine, self-isolation, social distance, masks, gloves, antiseptics and the like - are clearly insufficient. It takes time to create and organize the production of vaccines, and the infection spreads quickly, during this time the infection can develop from a local focus into an epidemic or even a pandemic. The example of the coronavirus pandemic continuing for the second year showed that the available antiviral drugs (RNA bases, reaferons, interferon inducers, virus replication inhibitors, etc.) are insufficient to provide the proper level of protection and treatment. To combat (prevention and treatment) with such infections, new types of drugs are needed that differ in mechanism of action from existing antimicrobial agents, and thus complement them. These drugs should have a complex antibacterial, antiviral and fungicidal activity, have an anti-inflammatory effect and generally increase the body's resistance to infection with pathogenic agents, that is, stimulate nonspecific immunity, both general and local (in particular, lysozyme activity). In this regard, nanostructured silver (nanosilver) preparations obtained using modern nanotechnology are promising. The purpose of this work is to develop and optimize synthesis methods, conduct tests of biological activity, toxicity, and show the capabilities of modern preparations of nanostructured silver (as well as some other metal nanoparticles - gold, platinum) when they are used in the fight against infectious diseases, including new and newly emerging infections. Our international scientific group, including researchers from Russia, Mexico, Spain and a number of other countries, has been developing and researching medical and veterinary drugs based on silver nanoparticles for a number of years. A large amount of work has been carried out to study the physico-chemical properties and biological activity of these drugs. They have been shown to be highly effective in the prevention and treatment of a number of infectious (both bacterial and viral) diseases of humans and farm animals and plants, including such as diabetic foot syndrome (diabetic ulcers of II and even III degree according to Wagner classification were treated), ENT infections, tuberculosis (including drug-resistant strains), Rift Valley fever virus, carnivorous plague virus (canine distemper), rotavirus infection, etc. The results of these studies have been published in more than 80 scientific articles, including more than 20 in Q1 and Q2 journals, a number of dissertations and patents. The crowning research can be considered the testing of a drug based on nanosilver at the General Hospital of Tijuana (Mexico), which currently specializes in the treatment of COVID patients. The conducted tests have shown the high effectiveness of this drug for the prevention of COVID-19 disease among the medical staff of the hospital (about 1200 medical workers + about 120 in the control group). These results were recently published in the journal Q1. Special attention is paid to the study of the cyto- and genotoxicity of nanosilver preparations being developed. Since nanosilver preparations, depending on the method of preparation, can significantly differ among themselves in terms of toxicological indicators and effectiveness, each nanosilver preparation claiming the status of a medicine or medical device must have its own scientific evidence base (dossier) on safety and effectiveness. It must be assumed that links and automatic transfer of data on properties from some nanosilver preparations to other nanosilver preparations, without additional experimental confirmation, are not authorized. Our studies have shown that various silver preparations, depending on the method of preparation and composition, can differ in toxicity by tens of times, which in most cases makes such preparations unsafe for humans. The substance we are developing and preparations based on it have an extensive scientific evidence base for safety and effectiveness. The results of toxicological studies have been published in leading journals (more than 80 articles, including more than 20 Q1 articles), dissertation abstracts (more than 10), patent applications and patents (more than 20) and scientific and methodological recommendations. All the results showed the non-toxicity of the nanosilver preparations being developed in therapeutic doses. However, despite the large amount of research already conducted, there are still a number of problems, the solution of which will significantly increase the effectiveness of the nanosilver preparations being developed, both in terms of the synthesis method and in the field of biological activity of these materials. The essence of nanotechnology can be expressed by the importance of work on atomic and molecular assembly of structures. From the same set of atoms and molecules, it is possible to create a sufficiently large number of different structures, which, accordingly, will exhibit different properties. The task is to create a stable structure with the necessary properties. Our previous studies have shown that in the treatment of various infectious diseases, optimization of the physico-chemical properties of the resulting preparations is required - the size of metal particles and complexes based on them, their morphology, ligand environment (type of stabilizer molecule), concentration, etc. Depending on these parameters, nanosilver preparations exhibit different biological activity, as well as toxicological properties. That is, in each specific case, optimization of the method of synthesis of a nanopreparation, as well as the method of its application (dosage, frequency, etc.) is required. Thus, it is imperative to study the general laws of the effect of the nanosilver synthesis method on its physicochemical (the size of metal particles and complexes based on them, their morphology, ligand environment, type of stabilizer molecule, concentration, etc.) and biological properties (including bactericidal, antiviral properties, cyto- and genotoxicity), which is planned in this project. The main direction of work in this project will be related to the optimization of methods for the synthesis of silver and other metal nanoparticles and the study of their physico-chemical properties. Since within the framework of this project it is impossible to conduct full-fledged clinical trials of drugs on humans, the biological activity of nanometallic systems will be determined in vitro, as well as on animals (mice, pigs, cattle, birds) infected with a number of pathogens (E.coli, Salmonella enteritidis, P.auregenosa, Streptococcus Proteus, etc.). In addition, despite the fact that the bactericidal properties of silver have been known since Phoenician times, the mechanism of biological activity of silver, both ionic and nanometallic (colloidal), has not yet been established. There are more than 10 different hypotheses that have not yet received sufficient evidence. Our previous studies of nanosilver and nanosilver catalysts in liquid-phase reactions of organic compounds suggest the catalytic nature of the mechanism of biological action of nanosilver. Part of the work will be devoted to this topic. A comparative study of the physicochemical properties and biological activity of nanoparticles of silver - gold and platinum analogues under comparable conditions is also of interest. In the literature there is information about their activity in the treatment of arthritis, arthrosis, cancer. A comparison of the properties of these nanometallic particles will allow us to better understand the possible mechanism of the influence of nanometallic synthesis methods on their structure and activity.

Expected results
This project is a continuation of a long cycle of work by an international scientific group on the creation of highly effective medicines of a new generation. - The influence of the method of synthesis of nanosilver preparations on their physico-chemical properties will be studied - the size of metal particles and complexes based on them, their morphology, ligand environment, type of stabilizer molecule, concentration, etc. - The conditions for the synthesis of nanometallic preparations will be optimized depending on the target parameters of the structure and morphology of the resulting nanoparticles. Critical parameters of the synthesis process and storage conditions affecting the stability of the resulting nanosystems will be established. - The study of the antibacterial and antiviral activity of the substance will continue, its anti-inflammatory effect will be evaluated. The studies will be performed both in vitro and on animals. For initial testing, a supported 3T3L1 line of embryonic fibroblasts will be used as normal cell cultures. Breast cancer (MDA-231), prostate cancer (PC-3) and leukemia lines (Jurkat) will be used as tumor cell lines. The study of cytotoxic effects and cell proliferation will be carried out using colorimetric tests (MTT, resazurin test). The assessment of cell morphology will be performed by optical light and fluorescence microscopy (AxioVertA1 microscope, Zeiss, Germany). The study of the mechanisms of cell death will be carried out by the cytofluorimetric method using sets of fluorescent dyes (Cytoflex, Beckman Coulter, USA). - Studies of the bioactivity of nanosilver preparations on laboratory and target animal species (mice, pigs, cattle, poultry) infected with a number of pathogenic microorganisms (E.coli, Salmonella enteritidis, P.auregenosa, Streptococcus Proteus, etc.) will continue. - Work will continue on solving a number of fundamental problems associated with nanosilver preparations. Thus, a thorough study of the cyto- and genotoxicity of these drugs is necessary, since reports of silver toxicity often appear in the literature. - Studies of the antiviral activity of nanosilver on models of coronavirus and paramyxovirus infections of chickens will be conducted; analysis of the viral load of IBV and NDV viruses in the intestine and lungs by real-time PCR, analysis of the expression level of the Muc5AC, Muc2, IL18, CXCR4, IL10, IL6, NFkB genes. The distributions of viral agents in intestinal and lung tissues were studied by immunofluorescence microscopy - Preclinical trials of nanosilver preparations will be continued in the treatment of a number of infectious diseases of humans and animals - diabetic foot syndrome, cow mastitis, cancerous tumors, etc. - Studies will be conducted under comparable conditions of the physicochemical properties and biological activity of nanoparticles of silver - gold and platinum analogues. A comparison of the properties of these nanometallic particles will allow us to better understand the possible mechanism of the influence of nanometallic synthesis methods on their structure and activity. - An assessment of the possible catalytic nature of the mechanism of biological action of nanosilver will be carried out. Based on the results of the conducted research, it is planned to develop recommendations on the use of nanosilver preparations for the treatment and prevention of a number of infectious diseases of animals and humans, for the further production and distribution of new drugs, including abroad (in particular, in Latin America). The results of preliminary tests show that the synthesized drugs will exceed domestic and foreign analogues in terms of basic medical and economic characteristics.

Annotation of the results obtained in 2022
1. Physico-chemical properties of nanosilver preparations (AgNPs) have been investigated. AgNPs are mostly spherical, with an average size of 33.3 ± 5.6 nm, as determined by HR-TEM. DSC-TGA results show 18.8% coating agent and 1.2% metallic silver. UV-vis analysis showed plasmon surface resonance at 409 nm. The hydrodynamic diameter summing the diameter of a metallic silver nanoparticle and a hydrolyzed protein coating is determined using DLS and is 165.5±105 nm, and ζ is a potential of 2.3±4.7 mV. 2. The results obtained in this work showed that the drug AgNPs BioArgovit has selective antiproliferative and antiparasitic activity without signs of cytotoxic, genotoxic or toxic side effects in vitro for healthy systems. These results open up new opportunities in the development of selective, safe and effective AgNPs drugs for the treatment of cancer and parasitic diseases with a significant reduction in side effects. All the results of the toxicological assessment indicate category 5 (practically non-toxic) of the globally harmonized system of classification and Labelling of Chemicals 3. The high antiviral activity of Argovit C has been shown in organism models (chickens infected with coronavirus (IBV)). Nanoparticles stabilized with polyvinylpyrrolidone have the highest activity in comparison with Argovite C, Bioargovite and Argovite stabilized with dimethyl sulfoxide, which creates even more interesting prospects for studying antiviral activity in vivo. The lowest antiviral activity was observed in nanosilver preparations using DMSO and ascorbic acid as a stabilizer. The activity of Argovite preparations containing polyvinylpyrrolidone was the highest and persisted to a concentration of 1 mcg/ml. 4. The high antiviral activity of silver nanoparticles was shown on a model of paramyxovirus infection in mice. 5. The effectiveness of the combined use of nanosilver and antibiotics in the treatment of Staphylococcus aureus (cow mastitis) has been shown. Thus, treatment of AgNPs Argovit-C mastitis can partially restore the activity of antibiotics against S. dysgalactiae and S. aureus, while, on the contrary, treatment with antibiotics such as Spectromast LC and Lactobay increases the resistance of bacteria to antibiotics. The results of this work strengthen the hope that in the future the use of AgNP as efflux pump inhibitors will restore the activity of antibiotics and, thus, preserve a wide range of antibiotics on the market. 6. The use of AgNP in the treatment of carnivorous plague has led to a sharp increase in the proportion of dogs that recovered without consequences, compared with dogs treated without AgNP. The available data suggest that AgNPs therapy can be considered as a targeted treatment of dogs and a number of fur-bearing animals seriously affected by the carnivore plague virus. 7. The activity of silver nanoparticles against phytopathogens was studied on the example of apricot cultivation. The development of an effective method of apricot micropropagation and the study of the necessary conditions for the introduction of AgNPs into apricot sprouts, as well as the impact of its use on the parameters associated with proliferation, has begun.

Publications

1. Carlos R. Romo Quiñonez, Pindaro Alvarez-Ruiz, Claudio H. Mejia-Ruiz, Nina Bogdanchikova, Alexey Pestryakov, Carina Gamez-Jimenez, Wenceslao Valenzuela-Quinonez, Magnolia Montoya-Mejia, Eusebio Nava Pérez Chronic toxicity of shrimp feed added with silver nanoparticles (Argovit-4®) in Litopenaeus vannamei and immune response to white spot syndrome virus infection PeerJ, PeerJ, 2022, V. 10, article 14231 (year - 2022) https://doi.org/10.7717/peerj.14231

2. Cristian Pérez-Caselles, Nuria Alburquerque, Lydia Faize, Nina Bogdanchikova, Juan Carlos García-Ramos, Ana G. Rodríguez-Hernández, Alexey Pestryakov, Lorenzo Burgos How to Get More Silver? Culture Media Adjustment Targeting Surge of Silver Nanoparticle Penetration in Apricot Tissue during in Vitro Micropropagation Horticulturae, Horticulturae, 2022, Vol. 8, article number 855 (year - 2022) https://doi.org/10.3390/horticulturae8100855

3. Gastelum-Leyva F., Pena-Jasso A., Alvarado-Vera M., Plascencia-Lopez I., Patron-Romero L., Loera Castaneda V., Gandara-Mireles J.A. , Lares-Asseff I., Leal-Avila M.A., Almeida-Perez J., Bogdanchikova N., Pestryakov A., Almanza-Reyes H. Evaluation of the efficacy and safety of silver nanoparticles in the treatment of neurological and non-neurological distemper in dogs: a randomized clinical trial Viruses, Viruses, 2022, V. 14, article 2329 (year - 2022) https://doi.org/10.3390/v14112329

Annotation of the results obtained in 2023
1. The possibility of obtaining new types of modified nanosilver preparations was confirmed by adding small amounts (0.01 – 2%) of the modifier to base solutions of PVP and GJ. Ligands-complexing agents of ionic silver (ammonia, thiosulfate ion, citrate ion), soft reducing agents (glucose, one-, two-, triatomic alcohols, glucosamines, etc.) can be used as modifiers, 5 new samples of modified nanosilver preparations have been manufactured. To obtain a nanosilver preparation intended for the synthesis of catalysts for the oxidation of alcohols, it is proposed to use alcohols involved in oxidation processes as a modifying additive. Attention is drawn to the silver-containing sorbent and its potential for suppressing the processes of generalization and chronization of coronavirus infection in patients with COVID-19. 2. According to the small–angle X-ray scattering (MURR) method, the size of primary silver particles in Argovite is 1-6 nm. According to electron spectroscopy data, particles are aggregated in solution, the average size of aggregates ("secondary particles") is 10-20 nm. 3. Electron microscopic examination confirmed the presence of silver nanoparticles in the crystalline state in Argovit-C preparation. Ag NPS are present in an aggregated form, as well as in the form of single nanoparticles. The length of the aggregates is on average ~ 200 nm. The characteristic sizes of the overwhelming number of particles are in the range from 3 to 20 nm with an average size of 9.4 nm. LF Ag vary in shape, the predominant proportion of particles is spherical, some of them are faceted and represented by polyhedra with rounded vertices. High-resolution electronic images indicate a defect in the crystal structure of the fraction of larger Ag LPS. 4. The results showed that silver nanoparticles obtained by irradiation with polyvinylpyrrolidone and collagen hydrolysate are stable in solutions. Samples with various stabilizers were characterized by a wide average size distribution from 2 to 50 nm and a low zeta potential from -7.3 to +12.4 mV. All AgNPs drugs have shown dose-dependent cytotoxic effects on tumor cells. It was found that the particles obtained using a combination of polyvinylpyrrolidone/collagen hydrolysate have a relatively more pronounced cytotoxic effect compared to samples stabilized only with collagen or only with polyvinylpyrrolidone. The minimum inhibitory concentrations of nanoparticles were less than 1 microgram/ml for various types of tumor cells. 5. Neuroblastoma (SH-SY5Y) was found to be the most susceptible, and ovarian cancer (SKOV-3) was found to be the most resistant to silver nanoparticles. The activity of the AgNPs drug prepared from a mixture of PVP and PH studied in this work was about 50 times higher than the activity of other AgNPs drugs described in the literature. The results show that AgNPs preparations synthesized by an electron beam and stabilized with polyvinylpyrrolidone and protein hydrolysate deserve in-depth study for their further use in the selective treatment of cancer without harming healthy cells of the patient's body. 6. It was found out whether the changes in adhesive and anti-lysozyme activity caused by AgNPs contribute to the restoration of bacterial sensitivity to antibiotics. In vivo sampling was performed before and after treatment of cow mastitis with antibiotics or AgNPs. Isolates were identified and adhesive and anti-lysozyme activity were evaluated. These data were compared with the results obtained for pretreatment of control bacteria with AgNPs or antibiotics in vitro. The present study showed that treatment of AgNPs bacteria in vitro and in vivo: (1) reduces the ability of bacteria to adhere to cells, causing infection, and (2) reduces bacterial anti-lysozyme activity, thereby increasing the activity of lysozyme, a natural “antibiotic" present in living organisms. The data obtained contribute to the prospect of future use of AgNPs to restore the activity of antibiotics that are rapidly disappearing from the market. 7. The results of this work revealed two more reasons for the property of AgNPs to restore the sensitivity of bacteria to antibiotics. It was shown that the use of AgNPs reduced the adhesive activity of bacteria by 23-29% in in vitro and in vivo experiments, and the use of six antibiotics increased it by 35% in vitro and was practically unchanged (+2%) in in vivo experiments. The same trends were observed with respect to the anti-lysozyme activity of bacteria. After the use of AgNPs, the anti-lysozyme activity decreased by 11-23%; however, after the use of antibiotics, it increased by 8% (in vitro) and practically did not change (-2%) in in vivo experiments. These results showed that AgNPs: (1) reduces the ability of bacteria to bind to cells necessary for subsequent cellular infection, and (2) reduces bacterial anti-lysozyme activity. These are two effects that enhance the ability of AgNPs to restore bacterial sensitivity to antibiotics. Consequently, the results of our work showed that the treatment of AgNPs bacteria in vitro and in vivo reduces the ability of bacteria to develop resistance to drugs due to (1) a decrease in the ability of bacteria to get rid of antibiotics, (2) a decrease in their ability to adhere to cells, causing infection, and (3) a decrease in the anti-lysozyme activity of bacteria, thereby thereby increasing the activity of lysozyme, a natural “antibiotic" is present in living organisms. 8. Using fluorescently labeled forms of nanosilver, the distribution of nanosilver in the lungs and intestines of mice with aerosol application was studied. The rate of removal of silver nanoparticles from the respiratory tract, lungs and intestines was determined, recommendations were given on the frequency of use of nanosilver preparations to avoid excessive accumulation of silver in the body.

Publications

1. Cristian Pérez-Caselles, Lorenzo Burgos, Inmaculada Sánchez-Balibrea, Jose A. Egea, Lydia Faize, Marina Martín-Valmaseda, Nina Bogdanchikova, Alexey Pestryakov, Nuria Alburquerque The Effect of Silver Nanoparticle Addition on Micropropagation of Apricot Cultivars (Prunus armeniaca L.) in Semisolid and Liquid Media Plants, V. 12, No 7, 1547 (year - 2023) https://doi.org/10.3390/plants12071547

2. Evgenii V. Plotnikov, Maria S. Tretayakova, Diana Garibo-Ruíz, Ana G. Rodríguez-Hernández, Alexey N. Pestryakov, Yanis Toledano-Magaña, Nina Bogdanchikova A Comparative Study of Cancer Cells Susceptibility to Silver Nanoparticles Produced by Electron Beam Pharmaceutics, V. 15, No 3, 962 (year - 2023) https://doi.org/10.3390/pharmaceutics15030962

3. M. R. Garcia Garcia, N. Casares, L. A. Martinez Perez, E. J. Curiel, A. A. de Jesus Hernandez, N. Bogdanchikova, D. Garibo, A. G. Rodriguez-Hernandez, A. Pestryakov, S. C. Gamboa, L. F. Arias Ruiz, O. Torres Bugarin, P. Berraondo Silver nanoparticles induce a non-immunogenic tumor cell death Journal of Immunotoxicology, V. 20, No. 1, 2175078 (year - 2023) https://doi.org/10.1080/1547691X.2023.2175078

4. Nina Bogdanchikova, Maria Maklakova, Luis Jesús Villarreal-Gómez, Ekaterina Nefedova, Nikolay N. Shkil, Evgenii Plotnikov, Alexey Pestryakov Revealing the Second and the Third Causes of AgNPs Property to Restore the Bacterial Susceptibility to Antibiotics International Journal of Molecular Sciences, V. 24, No 9, 7854 (year - 2023) https://doi.org/10.3390/ijms24097854

5. - Можно ли лечиться серебром и не посинеть Коммерсантъ, - (year - )