Összes szerző

Kovács Kinga Dóra

az alábbi absztraktok szerzői között szerepel:

Balogh Anna
Label-free tracking of cell adhesion kinetics as a function of various parameters

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P35

Label-free tracking of cell adhesion kinetics as a function of various parameters

Anna Balogh^1,2, Kinga Dora Kovacs^1,2, Imola Rajmon^1,2, Inna Szekacs²,

Beatrix Peter², Robert Horvath²

¹Eötvös Lóránd University, Department of Biological Physics, Budapest, Hungary

² Nanobiosensorics Laboratory, Institute of Technical Physics and Materials Science, Centre for Energy Research

Most tissue cells cannot survive for more than a few hours without adherence, if we were able to prevent the adhesion of malignant cells, metastasis could be prevented [1] [2]. The data used for analysis was recorded by a surface sensitive, label-free, resonant waveguide grating based optical biosensor. Due to the force calibration of the optical signal provided by the sensor for individual cells, this technique is suitable for determining the force curves of a large number of cells [3]. To analyse the adhesion signals at the molecular level, we applied and developed different kinetic models. These models take into account relevant molecular parameters, such as dissociation and association rates, the two dimensional kinetic dissociation constant which describes the integrin-ligand binding strength [4].

We investigated the extent to which the adhesion kinetics of cells are affected by the surface density of the cells, the density of echistatin, and the effect of gold nanoparticles on cell adhesion. Echistatin is a potent inhibitor of αIIβ3, αvβ3 and α5β1 receptors [5]. Functionalized nanoparticles can penetrate into living cells, Peter et. al. demonstrated from the recorded kinetic adhesion data that the uptake of the functionalized nanoparticles is an active process [6]. Peter et. al. also investigated the role of glycocalyx components in the cellular uptake of nanoparticles [7]. Subjecting these results to further analysis, we developed procedures and codes that enable the study of molecular parameters influencing adhesion in living cells, in their real environment, without isolating the relevant molecules. Our results potentially open the way for further analysis of the kinetic data obtained from the adhering cells.

Acknowledgment

This work was supported by the National Research, Development, and Innovation Office (Grant Numbers: PD 134195 for Z.Sz, PD 131543 for B.P., ELKH topic-fund, "Élvonal" KKP_19 TKP2022-EGA-04 grants).

References

[1] Sudhakar A. History of Cancer, Ancient and Modern Treatment Methods. J Cancer Sci Ther. 1, 2 (2009). https://doi.org/10.4172/1948-5956.100000e2

[2] Lodish, H., Berk, A., Matsudaira, P., et al. Molecular Cell Biology. (2003)

[3] Sztilkovics, M., Gerecsei, T., Peter, B. et al. Single-cell adhesion force kinetics of cell populations from combined label-free optical biosensor and robotic fluidic force microscopy. Sci Rep 10, 61 (2020). https://doi.org/10.1038/s41598-019-56898-7

[4] Kanyo, N., Kovacs, K.D., Saftics, A. et al. Glycocalyx regulates the strength and kinetics of cancer cell adhesion revealed by biophysical models based on high resolution label-free optical data. Sci Rep 10, 22422 (2020). https://doi.org/10.1038/s41598- 020-80033-6

[5] Szekacs, I., Orgovan, N., Peter, B., et. al. Receptor specific adhesion assay for the quantification of integrin–ligand interactions in intact cells using a microplate based, label-free optical biosensor. Sensors and Actuators B: Chemical, 256 (2018). https://doi.org/10.1016/j.snb.2017.09.208

[6] Peter, B., Lagzi, I., Teraji, S., et. al. Interaction of Positively Charged Gold Nanoparticles with Cancer Cells Monitored by an in Situ Label-Free Optical Biosensor and Transmission Electron Microscopy. ACS Appl. Mater. Interfaces 10, 32 (2018). https://doi.org/10.1021/acsami.8b01546

[7] Peter, B., Kanyo, N., Kovacs, K.D., et. al Glycocalyx Components Detune the Cellular Uptake of Gold Nanoparticles in a Size- and Charge-Dependent Manner. ACS Appl. Bio Mater. 6, 1 (2022). https://doi.org/10.1021/acsabm.2c00595

Farkas Enikő
Controlling Live Cell Adhesion through Characterization of Biofunctionalized Surfaces using Label-Free Biosensors

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P41

Controlling Live Cell Adhesion through Characterization of Biofunctionalized Surfaces using Label-Free Biosensors

Eniko Farkas¹, Kinga Dóra Kovács^1,3, Beatrix Peter¹, Attila Bonyár², Sandor Kurunczi¹, Inna Szekacs¹, and Robert Horvath¹

¹ Nanobiosensorics Laboratory, Institute of Technical Physics and Materials Science, Centre for Energy Research, Budapest, Hungary

² Department of Electronics Technology, Faculty of Electrical Engineering and Informatics, Budapest University of Technology and Economics, Budapest, Hungary

Biomaterial coatings that possess cell-repellent or cell-adhesive properties have a significant interest in medical and biotechnological applications [1-4]. However, conventional approaches lack in-depth analysis and quantitative comparison of these coatings for regulating adhesion, particularly for bacterial cell adhesion. Label-free Optical Waveguide Lightmode Spectroscopy (OWLS) can offer a solution for the detailed analysis of biomaterial coatings. OWLS biosensors detect the optical properties of the adhesive surface using evanescent waves with a penetration depth of 100-150 nm [5-7]. This surface-sensitive technique enables a thorough evaluation of biomaterial coatings for regulating adhesion. Uniquely, OWLS enables the in situ measurement of both the coating process and subsequent cell adhesion.

The present study utilizes the OWLS method for in-depth characterization of biomaterial surfaces with regard to bacterial adhesion. Initially, adhesion blocking biomaterials, namely bovine serum albumin, I-block, PAcrAM-g-(PMOXA, NH2, Si), (PAcrAM-P), and PLL-g-PEG, with varying coating temperatures, were screened. PAcrAM-P exhibited the best blocking capability against bacterial concentrations up to 10⁷ cells/mL. Subsequently, different immobilization methods, such as Mix&Go (AnteoBind) films, protein A, avidin-biotin based surface chemistries, and simple physisorption, were employed to captureEscherichia coli specific antibodies. Bacterial cell adhesion was then tested on immobilized antibodies with various blocking agents. The OWLS analysis allowed for the determination of the parameters of the applied agents by considering the kinetic data of adhesion, the surface mass density, and the protein orientation. Based on the experimental results, surfaces were created and tested for controlling both bacterial and mammalian cell adhesion. [8]

Acknowledgment

This work was supported by the "Lendület" (HAS) research program, the National Research, Development and Innovation Office of Hungary ((ERC_HU, VEKOP 2.2.1-16, ELKH topic-fund, "Élvonal" KKP_19 and KH grants, PD 131543 and TKP2022-EGA-04 –INBIOM TKP Programs financed from the NRDI Fund). This work was also supported by 77 Elektronika Ltd. by their supplying of antibodies and reagents.

References

[1] Frutiger A, et. al. (2021) Chem Rev 121: 8095–8160.

[2] Rigo S, et. al. (2018) Adv Sci 5: 1700892.

[3] Castillo-Henríquez L, et. al. (2020) Sensors 20: 6926.

[4] D’Agata R, et. al. (2021) Polymers 13:1929.

[5] Vörös J, et. al. (2002) Biomaterials 23: 3699–3710.

[6] Tiefenthaler K, et. al. (1989) J Opt Soc Am. B 6: 209–220.

[7] Saftics A, et. al. (2021) Adv Colloid Interface Sci 294: 102431–102433.

[8] Farkas E, et. al. (2022) Biosensors 12: 56.

Kovács Dóra Kinga
Nanoinjection of fluorescent nanoparticles to single live cells by robotic fluidic force microscopy

Aug 29 - kedd

12:15 – 12:30

Modern biofizikai módszerek

E13

Nanoinjection of fluorescent nanoparticles to single live cells by robotic fluidic force microscopy

Kinga Dóra Kovács^1,*, Tamás Visnovitz^2,3,*, Tamás Gerecsei¹, Beatrix Peter¹, Sándor Kurunczi¹, Anna Koncz², Krisztina Németh², Dorina Lenzinger², Krisztina V. Vukman², Péter Lőrincz⁴, Inna Székács¹, Edit I. Buzás^2,5,6**, Robert Horvath^1,**

¹ Nanobiosensorics Laboratory, Centre of Energy Research, ELKH, Budapest, Hungary

² Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary

³ Department of Plant Physiology and Molecular Plant Biology, ELTE Eötvös Loránd University, Budapest, Hungary

⁴ Department of Anatomy, Cell and Developmental Biology, ELTE Eötvös Loránd University, Budapest, Hungary

⁵ HCEMM-SU Extracellular Vesicle Research Group, Budapest, Hungary

⁶ ELKH-SE Translational Extracellular Vesicle Research Group, Budapest, Hungary

*,** equal contributions / **corresponding authors

Direct injection of fluorescent nanoparticles into the cytoplasm of living cells can provide new insights into the intracellular fate of various different fluorescently labelled biologically active particles. Here we used fluorescent nanoparticles to prove the feasibility of nanoinjection into single live HeLa cells by using robotic fluidic force microscopy (FluidFM). This injection platform offers the advantage of high cell selectivity and efficiency. We confirmed the successful injection of both GFP encoding plasmids and GFP tagged fluorescent nanoparticles to the cells by confocal microscopy. We were able track the nanoparticles in the living cells for 20 hours. The injected nanoparticles were initially localized in concentrated spot-like regions within the cytoplasm. Later, they were transported towards the periphery of the cells. Based on our proof-of-principle data, the FluidFM platform is suitable for targeting single living cells by fluorescently labelled biologically active particles and may lead to information about the intracellular cargo delivery at a single-cell level.

Madas Balázs
Metastatic potential of HeLa-cells does not increase directly after radiation exposure

Aug 29 - kedd

14:20 – 14:40

Orvosi biofizika és sugárbiológia

E15

Metastatic potential of HeLa-cells does not increase directly after radiation exposure

Balázs Madas¹, Kinga Kovács², Andrea Strádi³, Szabolcs Polgár⁴, Inna Székács⁵, and Róbert Horváth⁵

¹ Centre for Energy Research, Environmental Physics Department, Budapest, Hungary

² Centre for Energy Research, Nanobiosensorics Department, Budapest, Hungary

³ Centre for Energy Research, Space Research Department, Budapest, Hungary

While radiation therapy increases local tumor control, it remains controversial whether ionizing radiation increases the metastatic potential of cancer cells. One of the potential mechanisms of radiation-induced metastasis is the direct release of tumor cells into the circulation requiring the detachment of the cells. The objective of the present study was to directly measure how ionizing radiation affects the kinetics of cellular adhesion, especially its initial stage after cell attachment on a biomimetic surface.

For this purpose, an automatic irradiation facility with gamma-radiation from Cs-137 has been developed providing parallel irradiation opportunity of 96 wells of a biosensor microplate with different doses. The employed optical biosensor records the wavelength shift of reflected light from a nanostructured waveguide, being proportional to the cell adhesion strength. Absorbed doses were measured by thermoluminescent dosimeters (TLDs) in each well. As a model system, a cervical cancer cell line (HeLa) was studied.

Three different experimental setups have been used distinguished by the sequence of irradiation and cellular attachment to the surface. The wavelength shift as the function of time was measured for different absorbed doses. The maximum wavelength shift as the function of dose was also analyzed.

The results show that adhesion of HeLa cells is not affected by ionizing radiation in the first hours after irradiation. The result is independent of whether cells are exposed during the adhesion process, in suspension, or attached. This suggests that radiation therapy does not directly increase the metastatic potential of cancer cells by decreasing their adhesion.

The experimental setup can be used to quantify the effects of ionizing radiation on cell adhesion as the function of time at different absorbed doses. It has been shown that ionizing radiation does not affect the adhesion of HeLa cells in the first hours after exposure, while experiments with longer follow-up are required to see whether adhesion changes at later time points.

Péter Beatrix
Nanoparticle uptake of living cells with digested glycocalyx

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P21

Nanoparticle uptake of living cells with digested glycocalyx

Beatrix Peter^a, Nicolett Kanyo^a, Kinga Dora Kovacs^a,b, Viktor Kovács^a, Inna Szekacs^a, Béla Pécz^c, Kinga Molnár^d,Hideyuki Nakanishi^e, Istvan Lagzi^,,f,g, Robert Horvath^a

^a Nanobiosensorics laboratory, Institute of Technical Physics and Materials Science, Centre for Energy Research, Konkoly-Thege út 29-33, H-1121 Budapest, Hungary

^b Department of Biological Physics, Eötvös University, Budapest, Hungary

^c Thin Films Laboratory, Institute of Technical Physics and Materials Science, Centre for Energy Research, Konkoly-Thege út 29-33, H-1120 Budapest, Hungary

^d Department of Anatomy, Cell and Developmental Biology, ELTE, Eötvös Loránd University, Pázmány Péter stny. 1/C, Budapest, H-1117, Hungary

^e Department of Macromolecular Science and Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Kyoto 606-8585, Japan

^f Department of Physics, Institute of Physics, Budapest University of Technology and Economics, Műegyetem rkp. 3, Budapest H-1111, Hungary

^g ELKH BME Condensed Matter Research Group, Műegyetem rkp. 3, Budapest H-1111, Hungary

In biomedical imaging and targeted drug delivery, functionalized nanoparticles are widely used due to their penetration into living cells. The glycocalyx is a surface sugar layer of the cells, which presumably plays an essential role in any uptake process. However, its exact function in nanoparticle uptake is still uncovered. We in situ monitored the penetration of positively charged gold nanoparticles into adhered cancer cells with or without preliminary glycocalyx digestion. During the experiments, the components of glycocalyx of HeLa cells were digested by chondroitinase ABC enzyme. The measurements were performed by applying a high-throughput label-free resonant waveguide grating biosensor. The positively charged gold nanoparticles were used with different sizes (S, M, L). Negatively charged citrate-capped tannic acid nanoparticles, and other types of glycocalyx digesting enzymes were also applied in control experiments. The biosensor data confirmed the cellular uptake of the functionalized nanoparticles with an active process, which was verified by transmission electron microscopy [1,2]. Based on the findings we conclude that the components of gylcocalyx control the uptake process in size- and charge-dependent manner, and the possible roles of various glycocalyx components were highlighted.

Acknowledgements

This work was supported by the National Research, Development, and Innovation Office (Grant Numbers: PD 131543 for B.P., ELKH topic-fund, "Élvonal" KKP_19 TKP2022-EGA-04 grants).

References

[1] B. Peter, N. Kanyo, K. D. Kovacs, V. Kovács, I. Szekacs, B. Pécz, K. Molnár, H. Nakanishi, I. Lagzi, R. Horvath. Glycocalyx components detune the cellular uptake of gold nanoparticles in a size- and charge-dependent manner. ACS Applied Bio Materials, 2023.

[2] B. Peter, I. Lagzi, S. Teraji, H. Nakanishi, L. Cervenak, D. Zámbó, A. Deák, K. Molnár, M. Truszka, I. Szekacs, R. Horvath. Interaction of positively charged gold nanoparticles with cancer cells monitored by an in situ label-free optical biosensor and transmission electron microscopy. ACS Applied Materials & Interfaces, 2018.

Rajmon Imola
Single-cell adhesion measurements using fluidic force microscopy

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P23

Single-cell adhesion measurements using fluidic force microscopy

Imola Rajmon^1,2, Anna Balogh^1,2, Kinga Dóra Kovács^1,2, Inna Szekács² , Robert Horvath²

¹ELTE Eötvös Loránd University, Department of Biological Physics, Budapest, Hungary

²Nanobiosensorics Laboratory, Research Centre for Energy Research, Institute for Technical Physics and Materials Science, Konkoly-Thege u 29-33, 1120 Budapest, Hungary

Nowadays single-cell techniques are becoming valuable tools in the field of biology and biophysics. By investigating at a cellular level, we can better understand the cellular heterogeneity and the possible subpopulations in a tissue. This knowledge can bring novel applications and solutions to health and medicine. Fluidic Force Microscopy (FluidFM) is similar to atomic force microscopy (AFM), but uses a hollow, microfabricated cantilevers connected to a liquid reservoir and pressure controller system [1]. Through its precise force control, the cantilever is ideal to approach individual cells gently and reproducibly. Depending on the applications, the end of the cantilever can be different. For cell adhesion measurements 2-8 μm circular openings are ideal [2], while for live cell sampling and injections [3][4], so-called nanosyringes are used. These cantilevers have pyramidal tips with a 600 nm opening at their side. The cantilevers used for cell adhesion measurements can have different spring constants, which also determine some applications of the device. The short lecture will introduce a robotic version of the technique by presenting experimental results on the adhesive and mechanical properties of both cancerous and healthy cell types.

Acknowledgements

„Supported by the ÚNKP-22-2 New National Excellence Program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund.”

References

[1]: Li, M., Liu, L. & Zambelli, T. FluidFM for single-cell biophysics. Nano Res. 15, 773–786 (2022). https://doi.org/10.1007/s12274-021-3573-y

[2]: Sztilkovics, M., Gerecsei, T., Peter, B. et al. Single-cell adhesion force kinetics of cell populations from combined label-free optical biosensor and robotic fluidic force microscopy. Sci Rep 10, 61 (2020). https://doi.org/10.1038/s41598-019-56898-7

[3]: Chen, W., Guillaume-Gentil, O., Rainer, P.Y. et al. Live-seq enables temporal transcriptomic recording of single cells. Nature 608, 733–740 (2022). https://doi.org/10.1038/s41586-022-05046-9

[4]: Robert Horvath,Single-cell temporal transcriptomics from tiny cytoplasmic biopsies, Cell Reports Methods,Volume 2, Issue 10, 2022, 100319, ISSN 2667-2375, https://doi.org/10.1016/j.crmeth.2022.100319.