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Project titleImmobilized gold nanostars for high-efficient cell laser transfection

AffiliationInstitute of Biochemistry and Physiology of Plants and Microorganisms Russian Academy of Sciences,

Research area 04 - BIOLOGY AND LIFE SCIENCES, 04-209 - Biotechnology (including biological nanotechnology)

Keywordstransfection, culture cells, gold nanostars, cell optoporation, gene therapy

Annotation
The need to develop new drugs and approaches to the prevention of infectious diseases in livestock breeding is dictated by issues related to the growing need for food security and food independence of Russia. One of the most important problems in modern biomedicine is the intracellular delivery of bioactive substances. The systems based on the micro- and nanoparticles that encapsulate or adsorb nucleic acid molecules, and enhance membrane permeability resulting in enhanced transfection efficiency are one of the most promising and intensively studied to the date. Colloidal gold nanoparticles (AuNPs) are one of the most popular among the DNA nano-carriers. AuNPs is a promising class of nanomaterials for diagnostic purposes such as nucleic acids delivery due to the bio-inertness and low-toxicity of gold. It is worth investigating the optimal carriers for genetic material as well as the best delivery system while developing the transfection technology. The described elsewhere delivery systems include mechanical, chemical or physical methods. Recently, the optical transfection of cells using AuNPs has been actively studied in laboratory practice. The principle of the method is the enhanced energy, caused by laser irradiation of plasmon resonance nanoparticles localized near cell membranes, which leads to the short-term membrane penetrating activity for target molecules. Despite its high productivity and simplicity, the typical procedure contains a stage of incubating nanoparticles with cells, which can lead to undesirable effects associated with non-specific adsorption of nanoparticles, resulting in a decrease of transfection efficiency. Thus the proposal goal of the present project is the development of convenient, safe and efficient optoporation technology based on the immobilized Au nanostars monolayers in the bottoms of multi-well culture plates. The steps of nanoparticles incubation with the cells and subsequent washing of the culture medium from the exceed Au nanostars are eliminated in our approach thus giving significant reduce in operating time from several hours to several minutes, another advantage is that nanoparticles do not interfere with the assaying of transfection products. The increase in target biomolecules delivery efficiency will be achieved by careful optimization of the AuNSs parameters (monolayer density, particle size, etc.) and optical exposure modes (power, wavelength, time, etc.). The effectiveness of the proposed technology will be evaluated in comparison with the commercially available chemical transfecting agents in terms of the number of transfected cells and their viability by modern molecular genetic, microscopic, spectroscopic and cultural methods.

Expected results
Cell optoporation is a new promising strategy for intracellular delivery of nucleic acids (i.e. siRNA, DNA plasmids etc.). The main fundamental expected result of the present project is the development of a new high-effective and safe cell laser transfection technology based on the immobilized gold nanostars as exemplified on the plasmid DNA delivery into animal cells. The main expected result of the project for practical application: the proposed biomolecule delivery system will be more effective, low-cost and simple, as compared with the commercially available chemical transfecting agents in terms of the number of transfected cells and their viability by modern molecular genetic, microscopic, spectroscopic and cultural methods. The expected results of the project: 1. Novel technique of AuNSs monolayers immobilization on culture plastic as a special non-toxic and reusable surface coating for cells transfection will be developed for the first time, 2. Experimental scheme for animal cells laser transfection based on AuNSs immobilized on the culture plastic will be developed for the first time. 3. Comparative studies for estimation the efficiency of our laser transfection technology based on the immobilized AuNSs versus commercial chemical transfecting agents will be carried out. The proposed studies are actual, priority, comparable to the world level of scientific research, and they are ahead of similar foreign developments in this field of science in a number of positions. Taking into account the novelty of the present project, it is planned to present the main results in Russian and foreign publications (at least three publications indexed in the Web of Science or Scopus databases, at least three publications In the Russian index of scientific citation). Besides, the main results of the project will be presented on Russian and international scientific conferences. Based on the results obtained under the project, it will be possible to improve the work,the problem defined in the Strategy for Scientific and Technological Development of Russian Federation (approved by the Decree of the President of Russian Federation No. 642 of December 1, 2016 "On the Strategy for Scientific and Technological Development of Russian Federation"), which is part of the N4 direction named as: The transition to a highly productive and environmentally friendly agro- and aquaculture, the development and implementation of systems for the rational use of chemical and biological protection of agricultural plants and animals, storage and efficient processing of agricultural products, the creation of safe and high-quality, as well as functional foodstuff.

Annotation of the results obtained in 2018
The efficacy of the developed technology for in vitro cell plasmonic optoporation on monolayers of gold nanostars was quantified. HeLa cells were grown directly on Au nanostar layers, after which they were subjected to continuous-wave 808 nm laser irradiation. An Au monolayer density ~ 15 µg/cm2 and an absorbed energy of about 15-30 J (irradiation intensity, the irradiated area surface and the irradiation time were 1 W/cm2; 0.5 cm2, and 150 seconds, respectively), the operating temperature range during the laser exposure about 42-45 ° C were found to be optimal for optoporation. Propidium iodide molecules were used as model penetrating agent. The plasmid vectors carrying the fluorescent proteins genes with intracellular (pGFP) or extracellular (pCMV-GLuc 2) expression in mammalian cells were chosen As model cargo molecules. A preparative amount of plasmids was obtained using molecular cloning technology in E. coli cells, followed by selective clone selection and maxi-prep plasmid DNA extraction. The transfection efficiency evaluated using fluorescence microscopy for HeLa cells transfected with pGFP under optimized optoporation conditions (95±5%) was similar to the efficiency of TurboFect. The technique’s efficiency (295±10 relative light units, RLU), demonstrated by transfecting HeLa cells with the pCMV-GLuc 2 control plasmid, was greater than that obtained by transfection of HeLa cells with the TurboFect agent (220±10 RLU). The cell viability in plasmonic optoporation (92±7%), too, was greater than that in transfection with TurboFect (75±7%). The main results of the research were published in two articles in the first quarterly issues (Q1), and presented on the international conference on the project topic. In addition, the materials of the project, were prepared to a press release based on popular science articles, and a public popular science lecture was read. Thus, the new method of intracellular delivery of genetic structures developed in this project is a simple and convenient system with high parameters of cell efficiency and survival. We hope that the results of the project can be in demand in laboratory and clinical practice, as the basis of new developments in the field of cell engineering, gene therapy, molecular diagnostics, pharmacology, etc., aimed at switching to personalized medicine and improving the quality of the human health.

Publications

1. Pylaev T., Vanzha E., Avdeeva E., Khlebtsov B., Khlebtsov N A novel cell transfection platform based on laser optoporation mediated by Au nanostar layers Journal of Biophotonics, 11, статья № e201800166 (year - 2018) https://doi.org/10.1002/jbio.201800166

2. Pylaev T.E., Avdeeva E.S., Khlebtsov B.N., Zakharevich A.M., Khlebtsov N.G. A novel centrifuge-based approach for tunable 2D layering of plasmonic nanoparticles Proc. SPIE ,Saratov Fall Meeting 2018: Computations and Data Analysis: from Nanoscale Tools to Brain Functions, 11067 (year - 2019) https://doi.org/10.1117/12.2522135

3. Vetchinkina E., Loshchinina E., Kupryashina M., Burov A., Pylaev T., Nikitina V. Green synthesis of nanoparticles with extracellular and intracellular extracts of basidiomycetes PeerJ, Вып. 7, № статьи e5237 (year - 2018) https://doi.org/10.7717/peerj.5237

4. Pylaev T., Avdeeva E., Khlebtsov B., Khlebtsov N Design of a novel gene delivery system based on Au nanostar layers upon laser irradiation International conference on nanomedicine and nanobiotechnology – 2018, Rome, Italy, 26-28 Sept. 2018., - (year - 2018)

5. - Ученые из России создали золотой "нано-шприц" для генной терапии РИА Новости, РИА Новости, Москва, 19 февраля 2019 г. (year - )

6. - Золотые нанозвезды помогут доставлять вещества в клетку Индикатор.ру, Индикатор.ру, Химия и науки о материалах, 19 февраля 2019 г. (year - )

7. - Золотые нанозвезды и лазер: генная пушка, бьющая точно в цель Научно-популярный лекторий "Око хокинга", Видеозапись открытой лекции Пылаева Т.Е. научно-популярного лектория "Око Хокинга" (year - )

8. - Ученые из России создали золотой «нано-шприц» для генной терапии Лента новостей Российского научного фонда, - (year - )

Annotation of the results obtained in 2017
We present a novel laser transfection approach that uses Au nanostar monolayers as plasmonic surfaces. The AuNS monolayer density, irradiation time, and working temperature were optimized, and the optoporation efficiency and cell viability were evaluated, in experiments with HeLa cells and the fluorescent dye propidium iodide as a model penetrating agent.

Publications

1. Khlebtsov B.N., Pylaev T.E., Khanadeev V.A., Bratashov D.N., Khlebtsov N.G. Quantitative and multiplex dot-immunoassay using gap-enhanced Raman tags RSC advances, V. 7, P. 40834-40841 (year - 2017) https://doi.org/10.1039/C7RA08113H

2. Pylaev T.E., Vanzha E.V., Khlebtsov B.N., Khlebtsov N.G. A novel cell transfection platform based on laser optoporation mediated by Au nanostar layers Journal of biophotonics, - (year - 2018)

3. Pylaev T.E., Fomin A.S., Vanzha E.V., Staroverov S.A., Khlebtsov N.G. Polyethyleneimine-entrapped gold nanoparticles as a potential delivery system for DNA vaccines against African swine fever virus Saratov Fall Meeting – 2017, Int. Symp. “Optics and Biophotonics”, Saratov, Russia, 26-29 Sept. 2017, - (year - 2017)

4. Vanzha E.V., Pylaev T.E., Khlebtsov N.G. Оптопорация клеток на основе иммобилизованных золотых нанозвезд: оптимизация процедуры и определение токсичности Объединенный научный форум «XII чтения памяти академика Ю.А. Овчинникова», «VIII Российский симпозиум «Белки и пептиды»: науч. тр. Москва, 18-22 сент. 2017 г. // Acta Naturae. Спецвып, Спецвыпуск. С. 176. (year - 2017)

5. Vanzha E.V., Pylaev T.E., Prilepskii A.Yu., Khlebtsov B.N., Khlebtsov N.G. Immobilized gold nanostars for high-efficient laser transfection of animal cells and its application for gene delivery Saratov Fall Meeting – 2017, Int. Symp. “Optics and Biophotonics”, Saratov, Russia, 26-29 Sept. 2017, - (year - 2017)