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Project titleHybrid nanomaterials for photopharmacology - luminescent nanoparticles@functionalized phosphonates with photo-controlled bioactivity

AffiliationFederal State Budgetary Educational Institution of Higher Education "Saint-Petersburg State University",

Research area 03 - CHEMISTRY AND MATERIAL SCIENCES, 03-602 - Chemistry of new organic and hybrid functional materials

KeywordsPhotopharmacology, hybrid nanomaterials, photo-switching of bioactivity, functionalized phosphonates, luminescent nanoparticles

Annotation
The project aims in creation of a hybrid material based on luminescent nanoparticles (NPs) and new functionalized phosphonates (for the first time synthesized by the authors of the project) that are immobilized on the surface of NP. The functionalized phosphonates proposed in the project are unique because they simultaneously exhibit biological activity – the anticholinesterase effect, and demonstrate a change in the inhibition of cholinesterase group enzymes upon resonant optical irradiation. As luminescent nanoparticles, nanoparticles of binary oxides doped with rare earth ions will be used. NPs are multifunctional, because, firstly, they are the host for the immobilization of new synthesized phosphonates, and secondly, they can be used for luminescent visualization of the localization, including biological organs and tissues. The proposed hybrid nanomaterial is of interest for photopharmacology and the implementation of a new approach to the treatment of diseases associated with impaired activity of cholinesterase enzymes. In particular, the proposed hybrid nanomaterial will provide localized drug effects on organs and tissues due to the photo spatio-temporal control of the biologically active substance (functionalized phosphonates) and visualization of the area of its therapeutic effect due to luminescence of nanoparticles. The introduction of the proposed hybrid systems into medical practice will allow in the future to solve the problem of the uncontrolled in time and space of the activity of drugs (including outside the body), will ensure the minimization of side effects and negative effects on healthy organs, and also exclude the accumulation of biologically active substances in the environment. In the course of research on this project, fundamentally new results will be obtained in the study of the chemistry of chloroacetylene phosphonates, as well as in studies of the biological activity (anticholinesterase activity, general toxicity) of phosphorus-functionalized systems. The effect of optical radiation of various spectral ranges on the biological activity of new functionalized phosphonates will be studied; the optimal characteristics of laser radiation (wavelength, intensity) and threshold values of the laser exposure dose for changing the biological activity of the proposed class of phosphonates will be established. The result of the project will be the synthesis of hybrid nanostructures, which are a combination of luminescent nanoparticles based on metal oxides doped with REI (LaVO4/Gd2O3: Tb3+/Eu3+) and new phosphonate compounds immobilized on their surface; study of the biological activity of hybrid nanoparticles and their toxicological characteristics under various conditions of optical irradiation; as well as the study of visualization of the area of localization of hybrid materials in biological test systems.

Expected results
As a result of the project, new photopharmacological agents will be developed - hybrid nanomaterials that simultaneously have 3 functions - luminescence, biological activity and the ability to change the value of biological activity as a result of optical irradiation. The combination of oxide nanoparticles doped with rare-earth ions (luminescence) in the synthesized hybrid nanomaterials and functionalized phosphonates immobilized on their surface (the presence of biological activity and the ability to change it under optical irradiation) will make it possible to create multifunctional objects for photopharmacology. New functionalized phosphonates will be synthesized, their physicochemical properties will be studied, and general toxicity will be determined. Their biological activity will be investigated with the determination of IC50 by the example of inhibition of cholinesterase activity. The change in the biological activity of functionalized phosphonates will be studied depending on the conditions of laser exposure. The synthesis of luminescent oxide nanoparticles will be carried out and their composition will be optimized to achieve a high luminescence intensity. A technique for immobilizing target functionalized phosphonates on the surface of luminescent oxide nanoparticles will be developed. A theoretical and experimental study of the biological activity of inhibiting the cholinesterase activity of new functionalized phosphonates, hybrid nanomaterials based on them, as well as its changes as a result of laser exposure will be carried out. The toxicological properties (general toxicity) of functionalized phosphonates and hybrid nanomaterials will be investigated on selected biological test organisms. The possibility of visualizing the localization area of hybrid nanomaterials in biological test organisms will also be studied. Hybrid materials of such functionality are of key interest for the medicine of the future, due to the possibility of creating on their basis an individual approach (precise selection of the dose, area and duration of the therapeutic effect) to the treatment of severe diseases associated with neurodegenerative disorders in the human body. The main scientific significance of the project lies in the development of compounds of a group of new functionalized phosphonates that have both biological activity and the ability to change the value of bioactivity as a result of exposure to laser radiation, as well as the creation of hybrid nanostructures based on them, allowing visualization of the area of therapeutic exposure. The key feature of the proposed functionalized phosphonates is their significantly different biological activity in various photoinduced conformational states. Controlling biological processes with light is a non-invasive and highly targeted way to control the therapeutic effect. The light source acts on the light-switchable molecules, changing their specificity to the biological target. Thus, systemic toxicity for the patient and the environment is reduced due to the minimization of the therapeutic concentration of the drug due to its precise localization in the body and photocontrol of its activity. In addition, it is important to mention that unlike chemical agents commonly used to regulate biological processes, photons do not cause residual pollution, and light emission can be controlled by adjusting the wavelength and intensity. Photopharmacology can be useful for studying a large number of molecular targets, as it helps to selectively activate or inhibit certain enzymes, receptors and other mechanisms in the cells of biological organisms when exposed to light. Currently, photopharmacology is considered as a necessary tool in the research and treatment of one of the neurodegenerative pathologies of greatest interest for the development of new drugs - Alzheimer's disease (AD). AD is a neurodegenerative disease associated with the loss of cognitive functions in a progressive and irreversible way, as a result of which a person loses social and everyday skills, up to the loss of the ability to self-service, and thus drop out of society. For these reasons, more research is needed to find therapeutic drugs that target different molecular targets in specific ways that improve the quality of life for people with AD. Currently available examples of photopharmacological agents are a combination of 2 components (pharmacor and photoswitch). Pharmacors often lose their effectiveness as a result of conjugation with photoswitches. Compounds that simultaneously demonstrate the 3 declared functions (luminescence, biological activity and the ability to change the amount of biological activity as a result of optical irradiation) are currently absent. The development of such an approach is extremely in demand for solving the main modern socially significant problems, primarily in the field of medicine and ecology - (i) the search for pharmacological substances of a new principle of action, (ii) reducing the anthropogenic load on ecosystems and (iii) developing the principles of personalized medicine.

Annotation of the results obtained in 2022
The project aims at the development and creation of new compounds, on the basis of which it is possible to implement the concept of biological activity photoswitching. Such compounds can be used in the future as "smart drugs", the biological activity of which can be tuned by optical radiation. Such compounds, which change their bioactivity depending on the conformation that changes under the optical radiation, form the basis of photopharmacology, a rapidly developing interdisciplinary field that unites the efforts of scientists in the fields of biology, medicine, organic chemistry, materials science, and laser chemistry. The concept has proven to be fruitful and has already been shown to be effective in photoswitchable antimicrobial therapy, cancer therapy, ophthalmology, and more. In world practice, there are examples of the successful synthesis of a number of new compounds that demonstrate photo-induced structural changes and, as a result, modulation of their activity in relation to target tasks; some of these substances are already in preclinical studies. However, all of the demonstrated compounds consist of two components - a "photoswitch" and a biologically active substance - a "pharmacophore". Among all the studied classes of photochromic compounds, the leading position is occupied by azobenzenes due to the simplicity of their synthesis, "photoswitching" abilities (such as a high extinction coefficient and quantum yield). Traditional drugs e.g. antibiotics, are usually used as pharmacophores. The loss of the necessary activity of the drug (pharmacophore) upon conjugation with a photochromic compound is a serious problem of photopharmacology that requires the development of independent solutions for each combination of a photoswitch-pharmacophore. The latter circumstance determines the interest in the development of substances that have both biological activity and the ability to changes under the optical radiation. Within the framework of this project, a new family of photoactive substances is being developed, which simultaneously possess both biological activity and the ability to change the value of biological activity after optical exposure. The biological activity of functionalized phosphonates was studied using inhibition of butyrylcholinesterase. Enzymes of the cholinesterase group are present in various tissues of the body, such as the brain, blood, liver, gastrointestinal tract, neuromuscular synapses, which determines the importance of the synthesis of new compounds with anticholinesterase activity as drugs for the treatment of a wide range of diseases. As a result of the studies of the first year, a synthesis procedure was developed and a series of O-functionalized phosphonates was synthesized, the effect of the aliphatic chain and the possibility of the reaction occurring upon the introduction of saturated alcohols were studied. 7 compounds of functionalized phosphonates were created using the developed method. Further, the general toxicity of the obtained compounds was studied using several test objects, since different living organisms have different resistance to adverse factors: Daphnia magna Straus, green unicellular algae Scenedesmus quadricauda Brb. and paramecium Paramecium caudatum. According to the results of the complex biotesting, it was shown that compound 7 is the most toxic for daphnia and algae. The rest of the compounds demonstrate less toxicity for all the test systems considered. Using the principle of the greatest environmental safety, safe concentrations of the studied phosphonates were found for all test objects. Compound 7 has the lowest safe concentration (10-6 mM), and the remaining samples of phosphonates 3, 4, 5 and 6 showed a safe concentration of 10-5 mM. At the next stage, the synthesized functionalized phosphonates (3-7) were studied for the presence of biological activity towards inhibition of butyrylcholinesterase. A calibration dependence was built for each compound, and IC50 and inhibition constants were calculated. An analysis of the obtained data made it possible to determine the IC50 values lying within the micromolar concentration range (16 μM - compound 4 (the strongest inhibitor), 52 μM for compound 3, 60 μM for compounds 5 and 6, and the lowest value for compound 7 – 7000 µM. A theoretical study of functionalized phosphonates using the density functional theory showed that compounds 3-5, 7 exhibit two metastable states in addition to the main one. A qualitatively different dependence was noted for compound 4 with a branched (nonlinear) radical: the presence of branching radicals leads to the formation of a large number of metastable states with similar energies. Molecular docking showed that the binding of compounds 3 and 4 with butyrylcholinesterase (BuChE) occurs in different ways: in the first case compound 3 reveals van der Waals interactions with BuChE, while in the second case compound 4 forms hydrogen bonds. Information about project: Russia 24, channel Science "Volume #35 "How to turn on drugs with light". Science # 105" https://www.youtube.com/watch?v=qf545bHX2y4

Publications

1. A.V. Egorova, D.M. Egorov, N.O. Sonin, I.E. Kolesnikov, D.V. Pankin, A.A. Manshina, R.I. Baichurin Synthesis of a New Series of β-Chloro-β-phenylvinylphosphonic Acid Chloride Derivatives Russian Journal of General Chemistry, 92 (10) 2191–2196 (year - 2022) https://doi.org/10.1134/S1070363222100322

2. Ilya Kolesnikov, Daria Mamonova, Dmitrii Pankin, Gulia Bikbaeva, Anastasia Khokhlova, Anna Pilip, Anastasia Egorova, Vladislav Zigel, Alina Manshina Photoswitchable Phosphonate–Fullerene Hybrids with Cholinesterase Activity Photochemistry and photobiology, 2022 (year - 2022) https://doi.org/10.1111/php.13720

3. I. Kolesnikov, D. Mamonova , D. Pankin, A. Pilip , A. Egorova , and A. Manshina Meet new photo-pharmacological agents – functionalised phosphonates with cholinesterase activity Сборник трудов конференции «International Conference on Advanced Laser Technologies (ALT)», Сборник трудов конференции «International Conference on Advanced Laser Technologies (ALT)». 2022. №22. (year - 2022)

4. - Выпуск #35 "Как включать лекарства с помощью света". Наука # 105 Канал Наука, Россия 24, - (year - )

Annotation of the results obtained in 2023
The project is aimed at creating new compounds on the basis of which the concept of photoswitching biological activity can be implemented. Such compounds could be used in the future as “smart drugs” whose biological activity can be tuned using optical radiation. Compounds that change their bioactivity depending on their conformation, which changes under the influence of light, form the basis of photopharmacology, a rapidly developing interdisciplinary field that unites the efforts of scientists in the fields of biology, medicine, organic chemistry, materials science, and laser chemistry. The concept has proven fruitful and has already demonstrated effectiveness in photoswitchable antimicrobial, anticancer therapy, ophthalmology, etc. In world practice, there are examples of the successful synthesis of a number of new compounds demonstrating photoinduced changes in structure and, as a result, modulation of their activity in relation to target tasks; Some of these substances are already undergoing preclinical studies. However, all demonstrated compounds consist of two components – a “photoswitch” and a biologically active substance – a “pharmacophore”. Among all the studied classes of photochromic compounds, azobenzenes occupy a leading position due to the simplicity of their synthesis and “photoswitching” abilities (such as a high extinction coefficient and quantum yield). Traditional medicines – antibiotics, etc. – are used as pharmacological agents. However, the main problem hindering the development of photopharmacology is the loss of the necessary activity of the drug (pharmacophore) upon conjugation with a photochromic compound, which is a serious problem that requires the development of independent solutions for each photoswitch-pharmacophore combination. The latter circumstance determines the interest in the development of substances that simultaneously have biological activity and the ability to change it under the influence of light This project is developing a new family of photoactive substances that simultaneously have both biological activity and the ability to change the amount of biological activity after optical exposure. The biological activity of functionalized phosphonates was studied using the example of inhibition of butyrylcholinesterase. Enzymes of the cholinesterase group are present in various tissues of the body, such as the brain, blood, liver, gastrointestinal tract, and neuromuscular junctions, which determines the importance of the synthesis of new compounds with anticholinesterase action as drugs for the treatment of a wide range of diseases. During the second year of the project, a series of N-functionalized phosphonates (compounds 8–11) were synthesized. The resulting compounds were studied using NMR spectroscopy, mass spectrometry and FT-IR spectroscopy. To optimize photoexposure to synthesized (N,O)-functionalized phosphonates, studies were carried out to find the most suitable experimental parameters. These studies were carried out with compound 4, which is the most typical representative of the series and has the most pronounced properties of bioactivity and photoswitching. As a result of optimization, the following were determined: concentration of (N,O)-functionalized phosphonates – 10-3 M; laser wavelength – 266 nm; laser radiation power – 70 mW; The duration of laser exposure is 60 minutes. A study of the effect of laser radiation on the optical properties of a series of (N,O)-functionalized phosphonates using absorption spectroscopy showed that for all compounds, laser irradiation leads to a decrease in optical density. In addition to the decrease in optical density, a shift of the absorption band to short wavelengths was also observed. NMR and Raman spectroscopy showed that laser exposure leads to the appearance of an additional isomer in the synthesized compounds. It was found that laser-induced cis-trans isomerization occurs in O-functionalized phosphonates, and trans-cis isomerization occurs in N-functionalized phosphonates. Measurement of the biological activity of the synthesized functionalized phosphonates before and after laser irradiation showed that the greatest difference was shown by compound 4 - from 10% to 90% at a concentration of 10-5 M, the smallest difference was observed by compounds 6 and 7, which changed the biological activity at the same concentration of approximately 2 times – 18% → 34% (6) and 18% → 46% (7). It was found that irradiation with a laser at a wavelength of 325 nm for 60 minutes showed less change in the degree of inhibition of butyrylcholinesterase compared to irradiation at a wavelength of 266 nm. The synthesized compounds demonstrate fairly stable biological activity for a long time (up to 2 weeks) after laser irradiation. The synthesis of crystalline particles activated by rare earth metal ions was carried out using two modifications of the Pechini method: molten salt synthesis for the complex oxide LaVO4:Eu3+ and the foaming method for the simple oxide Gd2O3:Tb3+. The physicochemical and functional properties of the obtained concentration series of crystalline powders were studied using X-ray phase analysis, scanning and transmission electron microscopy, static light scattering and luminescence spectroscopy. As a result of the analysis of experimental results, LaVO4:Eu3+ 5 at.% powder was selected as the optimal sample for creating hybrid structures. This choice was due to the single-phase composition, the best particle size distribution and intense luminescence. The project implemented two methods for immobilizing phosphonates on the surface of luminescent oxide nanoparticles: physical sorption and covalent binding. To obtain a hybrid material, nanocrystalline luminescent particles LaVO4:Eu3+ 5 at.% (LEu) were chosen as a carrier and di(prop-2-yl)[(Z)-2-chloro-2- phenylethenyl]phosphonate (compound 4). Using physical sorption, the hybrid material was prepared by mixing a solution of compound 4 and a colloidal solution of LEu nanoparticles on a magnetic stirrer for 30 minutes at room temperature. The creation of a hybrid material using covalent bonding was carried out in two stages: the first stage is the application of a linker to the surface of luminescent particles; the second stage is phosphonate immobilization. The short-chain silane (3-Aminopropyl)triethoxysilane was chosen as a precursor for the linker. The results of preliminary experiments to study the biological activity and the possibility of photoswitching of the resulting hybrids showed that the most promising method for creating a hybrid material from the point of view of further use for photopharmacology is physical sorption. Based on the results obtained in year 2, a number of publications were published in the media: https://rscf.ru/news/release/preparat-vse-vklyucheno-pomozhet-v-lechenii-neyrodegenerativnykh-zabolevaniy/ https://spbu.ru/news-events/novosti/preparat-vse-vklyucheno-sozdannyy-v-spbgu-pomozhet-v-lechenii https://naked-science.ru/article/column/prepvklyucheno-pomozhet-v https://nauka.tass.ru/nauka/19061815 https://scientificrussia.ru/articles/preparat-vse-vkluceno-pomozet-v-lecenii-nejrodegenerativnyh-zabolevanij https://polit.ru/articles/pro-science/ps_photo/ https://poisknews.ru/farmaczevtika/farmakologiya/preparat-vse-vklyucheno-pomozhet-v-lechenii-nejrodegenerativnyh-zabolevanij/ https://indicator.ru/medicine/preparat-vse-vklyucheno-pomozhet-v-lechenii-neirodegenerativnykh-zabolevanii-19-10-2023.htm https://inscience.news/ru/article/russian-science/14595 https://spbu.ru/news-events/novosti/preparat-vse-vklyucheno-sozdannyy-v-spbgu-pomozhet-v-lechenii http://knvsh.gov.spb.ru/news/view/6186/ http://neuronovosti.ru/preparat-vse-vklyucheno-pomozhet-v-lechenii-nejrodegenerativnyh-zabolevanij/ https://vecherka-spb.ru/2023/10/19/uchenie-iz-spbgu-sozdali-preparat-dlya-lecheniya-dementsii-kotorim-mozhno-upravlyat-s-pomoshchyu-sveta https://sankt-peterburg-gid.ru/news/zdorove/preparat-vse-vklyucheno-sozdannyy-v-spbgu-pomozhet-v-lechenii-neyrodegenerativnyh-zabolevaniy.htm https://sanktpeterburg-info.ru/v-mire/kvantovye-tochki-povysili-effektivnost-lekarstva-ot-nejrodegenerativnyh-boleznej.html https://historymed.ru/news/178842/

Publications

1. Gulia Bikbaeva, Anastasia Egorova, Nikolai Sonin,Anna Pilip, Ilya Kolesnikov, Dmitrii Pankin, Roman Boroznjak, and Alina Manshina Vinyl Phosphonates as Photopharmacological Agents: Laser-Induced Cis-Trans Isomerization and Butyrylcholinesterase Activity ChemPhotoChem, e202300131 (year - 2023) https://doi.org/10.1002/cptc.202300131

2. Gulia Bikbaeva, Anna Pilip, Anastasia Egorova , Ilya Kolesnikov, Dmitrii Pankin, Kirill Laptinskiy, Alexey Vervald, Tatiana Dolenko, Gerd Leuchs and Alina Manshina All-in-One Photoactivated Inhibition of Butyrylcholinesterase Combined with Luminescence as an Activation and Localization Indicator: Carbon Quantum Dots@Phosphonate Hybrids Nanomaterials, 13, 2409 (year - 2023) https://doi.org/10.3390/nano13172409

3. - Препарат «все включено» поможет в лечении нейродегенеративных заболеваний Пресс-служба РНФ, - (year - )

4. - Препарат «все включено», созданный в СПбГУ, поможет в лечении нейродегенеративных заболеваний Новости СПбГУ, - (year - )

5. - Препарат «все включено» поможет в лечении нейродегенеративных заболеваний Naked Science, - (year - )

6. - Квантовые точки повысили эффективность лекарства от нейродегенеративных болезней Тасс Наука, - (year - )

7. - ПРЕПАРАТ «ВСЕ ВКЛЮЧЕНО» ПОМОЖЕТ В ЛЕЧЕНИИ НЕЙРОДЕГЕНЕРАТИВНЫХ ЗАБОЛЕВАНИЙ Информация взята с портала «Научная Россия» Научная Россия, - (year - )

8. - Препарат «все включено», созданный в СПбГУ, поможет в лечении нейродегенеративных заболеваний Правительство Санкт-Петербурга Комитет по науке и высшей школе, - (year - )

9. - Ученые из СПбГУ создали препарат для лечения деменции, которым можно управлять с помощью света Вечерний Санкт-Петербург, - (year - )