Spectroscopic and Microscopic Techniques for Nano-Probing, Modeling, and Recognition of Interactions Between Red Blood Cells and Vascular Endothelial Cells at the Molecular Level – KIT

The project aims to design and apply spectroscopic and microscopic methodologies for nano-scale probing, modeling, and recognition of interactions between red blood cells (RBCs) and vascular endothelial cells (ECs) at the molecular level.

A multi-modal analytical approach will be employed, combining:

• Fourier Transform Infrared Spectroscopy (FTIR) and nano-IR spectroscopy,

• Raman Spectroscopy (RS), including Surface-Enhanced Raman Spectroscopy (SERS) and Resonance Raman Spectroscopy (RRS),

• Atomic Force Microscopy (AFM) and Scanning Near-Field Optical Microscopy (SNOM),
  alongside complementary reference techniques.

The project comprises five main research tasks:

1. Nano-IR membrane probing – Development of a nano-IR-based methodology for analyzing biochemical alterations in intact membranes of RBCs and ECs, as well as in membranes of RBCs interacting with EC membranes.

2. SERS membrane interaction modeling – Creation of SERS-based analytical tools to study membrane changes and model interactions between metal surfaces, RBCs, and ECs.

3. AFM and RS characterization of microvesicles (MVs) – Nano-scale microscopic analysis of cell membranes and microvesicles from RBCs and ECs, including those produced during cell–cell interactions, with additional spectroscopic assessment of MV biochemistry.

4. RRS cross-talk tracking – Spectroscopic examination of RBC–EC communication mediated by nitric oxide (NO) molecules in both in vitro and ex vivo systems.

5. Multimodal interaction description – Integration of data obtained from nano-IR, SERS, AFM, RS, and RRS to correlate biochemical and structural profiles of RBCs and ECs and assess how RBC alterations influence EC function.

The project investigates single intact cells and interacting RBC–EC systems using both in vitro human cell models (human RBCs and human aortic endothelial cells – HAECs) and ex vivo approaches (cells isolated from murine models of atherosclerosis, diabetes, and heart failure).

In the initial phase, nano-IR spectroscopy will be applied to study membrane alterations in intact cells and isolated membranes under disease-related and age-related conditions. Subsequent experiments will focus on label-free SERS and tag-based SERS for the specific detection of adhesive proteins involved in RBC–EC interactions, employing metal nanoparticle tags.

Further analyses will focus on microvesicle-mediated intercellular communication, where MVs will be examined using AFM, RS, and RRS, supported by flow cytometry, morphological analysis, and ektacytometry. The final stage will explore RBC–EC cross-talk mechanisms, with emphasis on hemoglobin (Hb) adduct changes inside RBCs in contact with ECs, using RRS supported by EPR, UV-Vis spectroscopy, and blood gas analysis.

Ultimately, the multimodal interaction model developed in this project will provide a comprehensive molecular-level description of RBC–EC interactions, helping to predict vascular endothelial status and disease-related alterations based on spectroscopic and microscopic cellular profiles.