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Project titleDevelopment of "smart" theranostic nanodevices as promising agents for fighting antibiotic-resistant bacterial infections

Research area 03 - CHEMISTRY AND MATERIAL SCIENCES, 03-405 - Nanostructures and clusters. Supramolecular chemistry. Colloid systems

Keywordstheranostics; antibiotic-resistant bacteria; smart materials, nanoparticles, ligand-sensitive nanosystems; bacteriophages; antifouling coatings; N-heterocyclic carbenes

Annotation
Development of the new methods for fighting bacterial infections, particularly their drug-resistant forms, is one of the most important issues in modern healthcare that gains greater medical and social significance every year. For instance, numerous studies conducted worldwide demonstrate that purulent-septic infections are among the most frequent complications in patients undergoing hospital treatment, which prolong in-hospital stay and increase overall mortality. Moreover, the recently observed steady growth of infectious illnesses is also mainly associated with the spread of drug-resistant bacterial strains. It should be noted with particular concern that such pathogens are characterized by multiple mechanisms of intrinsic and acquired resistance to antibiotics, tolerance to antiseptics and detergents, drying and ultraviolet irradiation. The risk of transmission of these pathogens by hospital personnel and everyday objects is severed by their ability to form biofilms on biotic and abiotic surfaces, and subsequently by their persistence for prolonged periods in hospital environment. Despite the obvious increase of research devoted to new ways to fight with resistant bacterial infections especially by using natural enemies of bacteria such as bacteriophages and their highly specific and effective lytic enzymes, there is still a wide range of unresolved issues that make it difficult to use these entities for medication. In this regard, the search for solutions that synergistically combine the best features of several approaches is most justified. Theranostic nanoagents can be considered as candidates. This project is focused on development of "smart" nanostructures that not only join highly specific recognizing bioreceptors and efficient antibacterial agents in a single agent, but provide an array of fundamentally novel functions. Namely, the proposed approach would significantly improve the accuracy and specificity of recognition of biological targets by simultaneous analysis of several parameters of different nature. Within the project, systems that can analyze both surface markers of bacteria and soluble components of local environment (metabolites and other compounds) will be created. Such systems could become the central place in design of a new generation of highly selective drugs, which can concurrently analyze large amounts of parameters to accurately locate a target for its precise destruction and, simultaneously, are safe for normal cells. In addition, the use of nanostructures as agents to fight with bacterial pathogens provides unique opportunities for their highly effective destruction by adding functional properties, for example, by introducing nanoparticles with magnetic or photosensitive properties, as well as other agents capable of physically affecting a bacterial target. In combination with the above-described mechanism of specific recognition of a pathogen, such functional nanostructures may offer increased efficiency for bacterial destruction, e.g. by hyperthermia under the influence of a high-frequency magnetic field or generation of singlet oxygen. To achieve this extremely promising, but complex multidisciplinary goal, it is necessary to solve the following tasks: (i) Develop nanoagents specific to different bacteria and capable of transporting a number of antibacterial agents (for example, antibiotics, antimicrobial peptides, lytic enzymes, endolysins and nanoparticles). During the project, various bioligands will be applied as recognition components, including highly specific depolymerases that interact strictly with certain bacteria. To achieve high selectivity of binding to bacterial targets with low level of nonspecific interaction, the optimal composition, structure and colloidal and chemical properties of the nanoagents will be determined using extensive experience of the project team and, in particular, its leader, E. L. Kolychev, PhD (4 publications in JACS, IF = 13.858) in the field of synthetic organic and organometallic chemistry, including chemistry of N-heterocyclic carbenes, using self-organizing polymer layers with anti-fouling properties, using copper-free click-chemistry, highly-oriented functional layers, etc. (ii) Develop a system for analysis of local microenvironment around the nanoagent and control of its binding to bacterial targets. It is proposed to use an innovative biocomputing approach that allows producing nanostructures capable of performing mathematical calculations with biomolecules. The fundamental principles of this approach were developed at the Laboratory of Nanobiotechnologies of MIPT by one of the pioneers of this research filed (M. P. Nikitin, et al., Nature nanotechnology .- 2014.-V. 9.-N. 9.-P. 716-722). The laboratory is one of the recognized world leaders in the field of nanobiotechnology, which is confirmed by various awards (M. P. Nikitin was awarded by the President's Prize for Science and Innovation for Young Scientists in 2017, received the Second Prize of the Falling Walls Lab (Berlin, 2016) – the International competition for young innovators among more than 2400 research works from 50 countries; K. G. Shevchenko and M. P. Nikitin and co-authors were awarded the Second Prize at the Biosensors & Bioelectronics Award 2016, Gothenburg, Sweden). (iii) Demonstrate in practice the developed concept of the systems for fighting antibiotic-resistive bacterial infections. For this purpose, it is proposed to develop ligand-sensitive supramolecular computational structures capable of analyzing the concentration profile of various water-soluble markers (modeling cell metabolites) according to the laws of Boolean logic and implement binding with a target bacterium only in the case of a correct profile of markers. Such an approach will make the nanostructures much more selective and controllable. A separate scientific group of young scientists will be formed at the Laboratory of Nanobiotechnologies of MIPT for implementation of this particular clinically relevant application of the biocomputing concept. The head of the group will be a young specialist in the field of synthesis and modification of organic and organometallic compounds, Evgeny Leonidovich Kolychev, who has already received international recognition due to a number of works in high-ranking scientific journals (4 articles in JACS, European Union Marie Curie Intraeuropean Fellowship (Oxford, UK, 2014-2016)) . The experience and knowledge in the field of synthetic chemistry will be further supplemented by the skills of other Project team members who are highly qualified and experienced in solving multidisciplinary problems, including the fields of nanotechnology (PhD M. P. Nikitin), molecular biology (K. G. Shevchenko) and microbiology of bacteriophages (PhD A. V. Popova). This team provides unquestionably high probability of successful implementation of the complex and multidisciplinary tasks of the Project. Besides, the support of this Project will contribute to maintaining the leading international positions of our country in this research field, and will create prerequisites for demonstrating the real use of smart materials, which, at the moment, are at the fundamental rather than applied stage of research and development.

Expected results
The main outcome of the Project will be a significant advance in solving the global problem of fighting with antibiotic-resistant pathogens by developing a fundamentally new approach based on the use of theranostic nanostructures, which can independently and accurately identify a bacterial target and make a decision about its most effective destruction without any harm to the healthy cells. On the way of solving this complex multidisciplinary problem, it is necessary to elucidate a number of subtasks. The first step is obtaining nanostructures, the specificity of which to bacterial strains could be regulated by the presence of molecular markers. During the Project, the nanostructures based on the specially synthesized self-assembled nanoparticles and highly specific biomolecules will be created for precise identification, binding and eradication of bacteria or biofilms. Further, such structures will be programmed to "turn on" and/or "turn off" the ability to interact with bacterial targets. Such switching will be realized in the presence of certain small molecules, simulating, for example, the products of the vital activity of bacteria, which will further allow additional selection of bacterial targets from their metabolites. Such nanostructures will not only allow more accurate identification of bacterial targets, but will become the basis for the future agents capable of launching specific mechanisms of bacteria elimination and switching between targets depending on environmental factors. According to the authors’ opinion, a step-by-step solution of abovementioned problems will allow developing a fundamentally new approach to fighting with antibiotic-resistant bacterial pathogens, and also lay a foundation for solving other practical biomedical problems of high social significance.

Annotation of the results obtained in 2020
As part of the final year of the project implementation, the publication activity obligations were fully completed (1 publication in Q1 journals according to Scimago) and research on the development of a “smart” nanostructure for specific bacterial targeting under the control of low-molecular model ligands was successfully completed. A number of parameters responsible for the sensitivity and affinity switching of nanostructures have been refined and optimized, and quantitative and semi-quantitative methods for recording these parameters have been developed. For five different nanoparticle-immobilized phage depolymerases, the data on the rate of the bacterial biofilm recognition and its subsequent destruction within a relatively short time were statistically confirmed. The functionality of "smart" switching nanostructures that bind to bacterial cells when specific model ligands appear in their microenvironment have been demonstrated. An assessment of the sensitivity of bacterial targeting under the control of the model ligands was carried out. High specificity of targeting was noted in relation to both the ligands and the target bacterial strain. The possibility of creating multispecific nanostructures by coimmobilization of a cocktail of few depolymerases of different specificity, as well as ligand-sensitive agents capable of selective targeting of bacterial strains modulated by the appearance of the model marker, has been demonstrated. A way to further enhancing the bactericidal action of “smart” ligand-sensitive nanostructures based on phage depolymerases by embedding of additional antibacterial drugs (like silver nanoparticles) with subsequent modulation of their release under the influence of external chemical factors has been shown.

Publications

1. Artem A. Sizikov, Marianna V. Kharlamova, Maxim P. Nikitin, Petr I. Nikitin, Eugene L. Kolychev Non-viral locally injected magnetic vectors for in vivo gene delivery: a review of studies on magnetofection Nanomaterials, 2021, 11(5) (year - 2021) https://doi.org/10.3390/nano11051078

2. A.N. Kozyrina, A.A. Sizikov, A. Ringaci, A.V. Popova, D.V. Rogozhnikov, E.L. Kolychev Hybrid magnetic nanoparticles synthesized by the solvothermal method as promising agents for biomedical applications 2020 International Conference Laser Optics (ICLO), Proceedings, - (year - 2020)

3. A.N. Kozyrina, E.L. Kolychev, V.R. Cherkasov, M.P. Nikitin Химический синтез монодисперсных магнитных наночастиц с разнообразной морфологией на основе соединений железа для биомедицинских применений Перспективные Направления Физико-Химической Биологии и Биотехнологии. Сборник тезисов XXXII Зимней молодежной научной школы. 2020, Издательство: Институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова РАН (Москва), 2020, с. 130 (year - 2020)

4. E.L. Kolychev, A. Ringaci, A.A. Kotov, K.G. Shevchenko, M.P. Nikitin Plasmon resonance enhanced nontoxic nanoagents for in vivo detection of antibiotic resistant bacteria 2020 International Conference Laser Optics (ICLO), Proceedings, - (year - 2020)

5. - Реакция с Марса Найден новый способ синтеза наночастиц гематита для биомедицины Коммерсантъ, Наука от 28.05.2020 (year - )

Annotation of the results obtained in 2018
The main result of the first year of the Research Project is the successful development of approaches to design a monospecific nanoconstruct that recognize a given molecular target on the surface of model bacterial cells. First, the selection and synthesis of central microparticles suitable for the project objectives with optimal colloidal and chemical properties was carried out. Nanoparticles selection were based on the analysis of the published literature based on criteria such as biocompatibility, inertness and multimodality. All synthesized particles and particles obtained from commercial sources were characterized by modern physical and physicochemical methods of analysis. The following types of particles were selected and synthesized: Nanoparticles of hydrous iron oxide (III). A new method was developed for the synthesis of cubic particles of hydrous iron (III) oxide based on easily available starting materials (iron (III) chloride, ammonia and nitric acid). Surface coating with carboxymethyldextran has been shown to suppress nonspecific interactions with serum proteins such as albumin. Magnetic monodisperse nanoparticles of different nature. An effective method has been developed for obtaining superparamagnetic nanoparticles of different nature and size. The new method is versatile and allowing to synthesize both magnetite particles and ferrites of other metals, such as cobalt, manganese and copper in one-pot procedure with high yields of nearly monodisperse fraction of nanoparticles in the size range of 8-30 nm. Magnetic particles with core@shell structure. In order to produce magnetic particles with a surface suitable for versatile and selective modification, two methods of preparation of hybrid particles containing a magnetic core and a shell were successfully developed: 1) coating with silicon oxide and polysiloxanes and 2) coating with a layer metallic gold. Polymer particles. Particles of variable composition (based on polymers such as polystyrene and polymaleic anhydride) were prepared by the emulsion method. The focus of the synthesis was to prepare particles containing one or more magnetic core in the structure to ensure its multimodality. At the next stage of the study, the colloidal and chemical properties of central particles were studied and the effectiveness of covalent binding with various model receptors was evaluated. The bacterial strain Acinetobacter baumannii was chosen as a model system because it is one of the most dangerous agents of nosocomial infections, which is characterized by natural resistance to many antibiotics. The use of specific phage depolymerases was chosen as an effective approach to fight bacterial cells of A. baumannii and the biofilms formed by them. Enzymes that retained their activity after isolation and immobilization on a solid biochip surface as was identified by the method of spectral correlation interferometry were further used to develop methods for immobilizing bioligands on the surface of nanoparticles of various nature. The possibility of functionalization of gold nanoparticles with depolymerase, highly specific to A. baumannii strains, as well as other nonspecific proteins, was investigated. Conjugates based on nanoparticles of different nature were also obtained and a preliminary selection of several best conjugates was made. Studies have shown that gold nanoparticles are an excellent model for studying the basic processes of interaction of biofunctionalized (including depolymerase) nanoparticles with various bacterial models. At the same time, the use of magnetic nanoparticles with covalent immobilization of bioligands is a good basis for further experiments on the construction of “smart” anti-bacterial agents. A convenient and safe bacterial model was selected to develop methods for studying the specificity of the conjugates obtained and various signal detection methods were tested. For a more detailed study of the specificity of immobilized depolymerase and determination of methods for investigating their activity, we studied systems containing A.Baumannii and E.coli as candidates for model bacterial systems. To test the activity of immobilized phage depolymerase, a modified version of the classical bacteriological cell culture Petri dishes method was used as well as the original optical method for detecting the interaction of plasmon (gold) nanoparticles with bacteria. Based on the data obtained, we were able not only to show the preservation of the depolymerase activity of the enzyme immobilized on the surface of gold nanoparticles, but also to estimate the activity of depolymerase at its covalent immobilization on magnetic nanoparticles of different diameters. In addition to the methods described above, the original magnetic particle quantification method (MPQ) at combinatorial frequencies developed with the participation of team members of this Project were applied as an alternative method for detecting interactions involving magnetic nanoparticles. It was demonstrated that the combination of the quantitative MPQ method and the magnetic resonance imaging (MRI) method could give an adequate idea of the processes of interaction of nanoparticles with organs and tissues of a living organism, as well as evaluate their pharmacokinetic parameters. A significant part of the data obtained in the course of the project on MPQ studies was published in the high-rating Q1 journal (Zelepukin I.V. et al. (2018). Nanotechnology, 30 105101, IF = 3.404, https://iopscience.iop.org/article/10.1088/1361-6528/aafa3a/meta). Thus, the analysis of the obtained results shows the possibility of immobilization of phage depolymerase on the surface of nanoparticles while preserving the specificity of bacteria recognition of certain strains of antibiotic-resistant bacteria. Such recognition can have not only diagnostic significance, but also therapeutic use, since depolymerase retains its enzymatic activity and can be used for not only diagnostics, but also destroying the protective film of bacteria.

Publications

1. I.V. Zelepukin, A.V. Yaremenko, E.V. Petersen, S.M. Deyev, V.R. Cherkasov, P.I. Nikitin, M.P. Nikitin Magnetometry based method for investigation of nanoparticle clearance from circulation in a liver perfusion model NANOTECHNOLOGY, Том: 30, выпуск: 10, номер статьи: 105101 (8pp). (year - 2019) https://doi.org/10.1088/1361-6528/aafa3a

Annotation of the results obtained in 2019
Within the framework of the second year of the Project, the obligations on publication activity were fully fulfilled (3 publications, two of which are in Q1 journals according to Scimago). Development and preliminary testing of the simplest “smart” ligand-dependent antibacterial nanostructure sensitive to one model ligand were successfully completed. On the first step, suitable physical and biological methods were selected for studying interactions of various bacterial cultures with active conjugates of nanoparticles developed during the first year of the Project. Imaging flow cytometry and magnetic particle quantification (MPQ) cytometry were used to register interactions of the nanoparticles and qualitatively confirmed their specific and reverse character. Studies on the stability of depolymerases in the conjugates have shown that the depolymerase activity of the conjugates is due to these enzymes immobilized on the surface. Possibility of destroying a bacterial biofilm for further development of methods for fighting antibiotic-resistant infections was demonstrated. In addition, imaging flow cytometry and fluorescent immunoassay methods were used to evaluate the stability parameters of various polymer coatings on metal oxide nanoparticles. Effectiveness of these methods in investigation of nanocomposites stability was demonstrated on a model system. Polymer coating based on polyacrylic demonstrated optimal performance. After a thorough analysis of the central nanoparticles, efficient methods for synthesis of small nanoparticles of magnetite, hydrated iron(III) oxide and coinage metals were developed or optimized for use in nanocomposites as shielding particles. Thus, a method was developed for preparation of water-dispersible shielding nanoparticles from magnetite and metal ferrites with a carboxyl group terminated polymer surface layer. The known methods for the synthesis of small gold and silver nanoparticles were tailored and the optimal reagents for the stabilization of their aqueous solutions were selected, providing easy conjugation of their surface with biomolecules. Silver and gold particles stabilized by functionalized for rapid biomodification N-heterocyclic carbenes were obtained and their high stability in water solutions were demonstrated. In addition, thiosubstituted carboxylic acids were used for preparation of water-soluble nanoparticles of silver indium sulfide, which have luminescent properties in addition to the antibacterial activity of silver compounds. On the next step, on the basis of the model design “central microparticle - shielding nanoparticle”, a technique was developed for forming a layer of protective nanoparticles to shield immobilized bioligands from interaction with their molecular or biomolecular substrate. Several central particle/shielding particle combinations have been studied using the degree of blocking the activity of depolymerases against bacteria using a model input ligand, as a criterion. It was shown that the developed methods for screening depolymerases showed comparable efficacy and can be used to further development of “smart” anti-bacterial systems on their basis. Next, the operability of the nanoscale structures capable of responding to the model small molecules was tested. The key parameters for the functioning of nanocomposites were established. The behavior of individual elements of nanocomposites was studied in in vitro and in vivo conditions, including parameters such as the circulation time and their biodistribution by organs, as well as by their nonspecific toxicity. In particular, the nonspecific toxicity of hematite nanoparticles of various shapes and compositions was studied both in vitro (using the MTT test, imaging flow cytometry and fluorescence microscopy), and in vivo in mice using a biochemical blood test. As the result, it was shown that doping with a small amount of heavy metal does not affect the toxicity of hematite central nanoparticles. However, the geometric shape of the particles can cause their toxicity instead. Finally, in vivo magnetic resonance imaging showed an increase in the contrasting properties of hematite-based central nanoparticles with the introduction of a small amount of europium as a doping element. Using optical fluorescence tomography, the in vivo biodistribution the nanoparticles of hematite labeled with a fluorescent dye was analyzed in mice. In conclusion, the behavior of the nanocomposite magnetic components was studied by evaluating the parameters for removing particles from the bloodstream of laboratory animals using the MPQ method.

Publications

1. A.V. Lunin, A.A. Lizunova, E.N. Mochalova, M.N. Yakovtseva,V.R. Cherkasov, M.P. Nikitin, E.L. Kolychev Hematite Nanoparticles from Unexpected Reaction of Ferrihydrite with Concentrated Acids for Biomedical Applications Molecules, 25(8), 1984 (year - 2020) https://doi.org/10.3390/molecules25081984

2. A.V. Lunin, I.L. Sokolov, I.V. Zelepukin, I.V. Zubarev, M.N. Yakovtseva, E. N. Mochalova, J.M. Rozenberg, M.P. Nikitin, E.L. Kolychev Spindle-like MRI-active europium-doped iron oxide nanoparticles with shape-induced cytotoxicity from simple and facile ferrihydrite crystallization procedure RSC Advances, Vol. 10, pp. 7301-7312 (year - 2020) https://doi.org/10.1039/C9RA10683A

3. A. Ringaci, K.G. Shevchenko, V.R. Cherkasov, E.L. Kolychev MIL-100 coated nanoparticles as a potential tool for in vitro delivery of therapeutic agents Cell Death Discovery, Vol. 6, suppl. 1, p. 16, RPC 29 (year - 2020) https://doi.org/10.1038/s41420-020-0248-5