Atherosclerosis, as a process of hardening and narrowing of arteria, affects all people. However, not all plaques are dangerous. The discrimination between the stable atherosclerotic lesions from the dangerous ones is not possible by existing techniques. We introduce an in-vitromodel to target macrophages, which have a high abundance in vulnerable atherosclerotic plaques,by employing SERS tags.

V. Dugandžić // D. Cialla‐May 

Atherosclerosis is a disease in which the fatty deposits are accumulated in the arterial walls leading to their narrowing and hardening. It affects almost all humans in certain extent, being a leading cause of mortality in western world. The progression of atherosclerosis is often associated with the unhealthy life style, lack of physical activity and bad nutrition. The development of the lesions starts with the damage of the epithelial tissue followed by a cascade of biochemical processes resulting in formation of the fatty deposits. It is well known that the dangerous atherosclerotic plaques are characterized by the inflammation process. Therefore, the body’s defense mechanisms result with the recruitment of the white blood cells, with a goal to repair the tissue damage. The white blood cells, as a part of the immune response, get differentiated into macrophages, the cells which have a task to ingest the plaques, leading to formation of foam cells. The presence of macrophages in dangerous plaques might be used for the targeting of the dangerous atherosclerotic plaques with the purpose of their recognition. One of the promising techniques which may enable the early stage detection of the dangerous atherosclerotic plaques is surface-enhanced Raman spectroscopy (SERS) in combination with Raman endoscopy.

The strategy for detection of the dangerous atherosclerotic plaques using a SERS-based approach requires the delivery of the SERS-active plasmonic nanoparticles to the plaques, followed by the subsequent detection employing the Raman endoscopic probe. Atherosclerotic plaques in general lack specific markers which makes their targeting profoundly hard, however the abundance of macrophages within the dangerous plaques may be employed for the targeted delivery of SERS tags. It is well known that macrophages express a lectin type receptor on their surface which is specific for binding of mannose.  Therefore, the functionalization of SERS tags with mannose might be a possible targeting route.

To test the SERS-based approach for the detection of the dangerous atherosclerotic plaques via delivery of tags to the macrophages within the plaques, suitable SERS tags were designed. Branched gold nanoparticles were employed as a plasmonic core since they exhibit the SERS enhancement without need for aggregation.  The cores were further functionalized with a SERS label. A molecule containing cyano group, 1.4-phenylene diisocyanide (PDI), was employed as a SERS tag. An easy discrimination of the SERS signal from the Raman signal of cells and tissues was possible, since the specific cyano group vibration occurs at around 2150 cm-1, the spectral range where cells and tissues show no Raman signatures. In the next step the SERS tags were coated with a layer of silica to ensure biocompatibility and low toxicity of the labels. In the final step the mannose functionalization was introduced onto the surface of the SERS labels to achieve a selective targeting of macrophages.

Prepared SERS tags were further tested in vitro on macrophage cell lines to check for their interaction with macrophages. The estimation of the nanoparticle uptake by macrophages was performed employing the confocal Raman imaging (Fig. 1A). The results clearly show that the mannose functionalization results in a higher uptake of nanoparticles already after 30 minutes of incubation (Fig. 1B), with a dramatic increase of the uptake after 2 hours. In the case of nanoparticles without mannose functionalization smaller uptake was observed than in the case of the mannose functionalized nanoparticles. Furthermore, the stagnation of the uptake was observed already after 30 minutes.

The observed results indicate that the employment of mannose modified SERS tags for the detection of dangerous atherosclerotic plaques via targeting of macrophages might be a promising strategy for the discrimination of dangerous atherosclerotic plaques from the stable ones.

Funded by: BMBF, Carl-Zeiss-Stiftung