KIT

Projekt finansowany przez Narodowe Centrum Nauki w ramach konkursu „Sonata Bis – 10”.

Techniki spektroskopowe i mikroskopowe w nanosondowaniu, modelowaniu i rozpoznaniu interakcji
pomiędzy erytrocytami i komórkami śródbłonka naczyniowego na poziomie molekularnym.

Nr projektu: UMO- 2020/38/E/ST4/00197
Wartość projektu: 485.040,00 PLN
Wartość dofinansowania: 485.040,00 PLN
Okres realizacji projektu: 25.04.2022 – 24.04.2027

Kierownik projektu: dr Katarzyna Marzec

In this project, we will design and apply spectroscopic and microscopic methodologies for nano-probing, modeling and recognition of interactions between red blood cells (RBCs) and vascular endothelial cells (ECs) at the molecular level. Multi-modal approach includes the application of the combination of Fourier transform infrared spectroscopy (FTIR), particularly nano-scale infrared spectroscopy (nano-IR) as well as Raman spectroscopy (RS) including surface enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RRS) as well as atomic force microscopy (AFM) and scanning near field optical microscopy (SNOM) in combination with reference techniques. Implementation of the five tasks of the project will provide:

1) Nano-IR membrane probing: Design and application of nano-IR-based methodology for analysis of biochemical alterations in intact membranes of RBCs and ECs, as well as in the membranes of RBC interacting with EC membranes.
2) SERS membrane interaction modelling: Design and application of SERS-based methodology for analysis of membrane alterations in RBCs and modelling metal surface-RBCs and ECs-RBCs interactions.
3) AFM and RS in microvesicles (MVs) defining: Nano-scale microscopic surface analysis of cell membranes and MVs of RBC and EC, as well as of RBC membranes interacting with EC membranes, and spectroscopic analysis of biochemistry of released MVs.
4) RRS crosstalk tracking: Spectroscopic analysis of the RBCs-ECs interactions and communication via NO molecule in both in vitro and ex vivo systems.
5) Multimodal interaction description: Correlation of the results obtained with nano-IR, SERS, AFM, RS, and RRS-based approaches comprising characteristics of intact RBCs and ECs and the impact of RBCs’ alterations on ECs profile in vitro and ex vivo.
3) AFM and RS in microvesicles (MVs) defining: Nano-scale microscopic surface analysis of cell membranes and MVs of RBC and EC, as well as of RBC membranes interacting with EC membranes, and spectroscopic analysis of biochemistry of released MVs.
4) RRS crosstalk tracking: Spectroscopic analysis of the RBCs-ECs interactions and communication via NO molecule in both in vitro and ex vivo systems.
5) Multimodal interaction description: Correlation of the results obtained with nano-IR, SERS, AFM, RS, and RRS-based approaches comprising characteristics of intact RBCs and ECs and the impact of RBCs’ alterations on ECs profile in vitro and ex vivo.

The project assumes analysis of single intact cells as well as interacting RBCs and ECs for in vitro systems (human RBCs – human aortic endothelial cells – HAECs) as well as ex vivo approaches (RBCs isolated from murine models of atherosclerosis, diabetes and heart failure and ECs of isolated vessels). The first task is focused on nano-IR approach for membrane studies of intact RBCs and ECs in which the age-related and diseases-related alterations were induced in vitro. This will be followed by the ex vivo analysis of intact membranes of cells isolated from mice model of human diseases. At the end the nano-probing of alterations will be studied for RBC membranes in interaction with HAEC in vitro. In order to properly differentiate the information delivered from membranes of intact cells, the isolated membranes as well as cytosol will be also studied with the use of FTIR-ATR, RS, SNOM and classical techniques of analysis. The second task includes design and application of label –free SERS for analysis of the biochemical alterations in the ECs and RBCs membranes. Further on the specific recognition SERS based on tags will be developed for modelling of cells’ interactions. This includes design of the metal nanoparticles tags for analysis of chosen adhesive proteins important in RBCs-ECs interactions. Reference techniques include mainly flow cytometry to analyze the protein expression on the surface of cells and stains for RBC and EC glycocalyx. The third task is focused on the analysis of the cell-to-cell communication via MVs, which may serve as inter-cellular exchangers of information. MVs delivered by altered RBCs and altered ECs as well as due to their interaction will be analyzed in nano-scale on the cell membranes, with the application of AFM. Additionally, RS or RRS approach will be used to analyse the biochemical profile of MVs in supernatant. Reference techniques include MVs analysis with flow cytometry and analysis of their size, diameter and concentration as well as biochemical analysis, morphology and ektacytometry. The fourth task is focused on detection and differentiation of the initial triggers of cross-talk between the RBCs and ECs, focusing on the analysis of Hb adduct changes inside RBCs in contact with ECs. Such aspect will be studied with an appropriately designed RRS based approach with the support of other reference techniques (EPR, UV-Vis, blood gas analysis). Multimodal interaction description will reveal whether the biochemical profile of RBCs, delivered by spectroscopy/microscopy-based approaches, enables elucidation of the nature of RBCs interactions with ECs as well as prediction of the state of the ECs and vice versa.