Multiplexing Biosensor for the Detection of Extracellular Vesicles as Biomarkers of Tissue Damage and Recovery after Ischemic Stroke

Abstract

The inflammatory, reparative and regenerative mechanisms activated in ischemic stroke patients immediately after the event cooperate in the response to injury, in the restoration of functions and in brain remodeling even weeks after the event and can be sustained by the rehabilitation treatment. Nonetheless, patients' response to treatments is difficult to predict because of the lack of specific measurable markers of recovery, which could be complementary to clinical scales in the evaluation of patients. Considering that Extracellular Vesicles (EVs) are carriers of multiple molecules involved in the response to stroke injury, in the present study, we have identified a panel of EV-associated molecules that (i) confirm the crucial involvement of EVs in the processes that follow ischemic stroke, (ii) could possibly profile ischemic stroke patients at the beginning of the rehabilitation program, (iii) could be used in predicting patients' response to treatment. By means of a multiplexing Surface Plasmon Resonance imaging biosensor, subacute ischemic stroke patients were proven to have increased expression of vascular endothelial growth factor receptor 2 (VEGFR2) and translocator protein (TSPO) on the surface of small EVs in blood. Besides, microglia EVs and endothelial EVs were shown to be significantly involved in the intercellular communications that occur more than 10 days after ischemic stroke, thus being potential tools for the profiling of patients in the subacute phase after ischemic stroke and in the prediction of their recovery.

Keywords: Surface Plasmon Resonance imaging; biomarkers; biosensor; extracellular vesicles; stroke.

Figure 1

Experimental design. (A): Schematic representation of the experimental setting of the present study. (B). Schematic of the analysis performed by means of the SPRi biosensor: first the gold biochip is functionalized with antibodies or ligands specific for EVs from selected cell source; then, specific EV subfamilies are immobilized on the biochip thanks to tissue related markers; finally, the immobilized EVs are characterized with a secondary injection of antibodies conjugated with gold nanoparticles to enhance the SPRi signal, or synthetic ligands specific for surface molecules, in this case the TSPO ligand PK-11195. (C): Representative CCD differential image of the SPRi chip surface where the 12 families of ligands spotted on the biochip can be visually detected.

Figure 2

Stroke patients profiling. Box plots represent the quantification of soluble mediators of inflammation and regeneration by enzyme-linked immunosorbent assay on serum samples of control samples (CTRL; n = 16) and stroke patients (STROKE, n = 19), by means of ELISA plates or ELLA automated system. The results obtained for ICAM-1 (A), IL-6 (B), IL-10 (C), TNFα (D), Leptin (E), Fas (F), BDNF (G), VEGFR2 (H), Klotho (I) are reported. Mann–Whitney test was applied to all measurements to compare the distribution of the two experimental groups. The p-value is reported when the two distributions resulted statistically significant (p < 0.05).

Figure 3

Extracellular Vesicle characterization. (A): Box plot reporting the EV concentration obtained by NTA analysis on EV preparations from healthy controls (CTRL; black) and stroke patients (STROKE; red). Each dot represents the mean value obtained for each subject after five acquisitions, whereas X represents the mean value for each group. (B): Box plot reporting the μg of proteins per particles obtained for each EV preparations from healthy controls (CTRL; black) and stroke patients (STROKE; red). The value was obtained dividing the μg of proteins measured by BCA assay for the concentration of particles obtained by NTA. Each dot represents the mean value obtained for each subject, whereas X represents the mean value for each group. (C): Representative TEM image obtained from the analysis of EV preparations from healthy controls and stroke patients. Black bar: 200 nm. (D) Western Blot analysis performed on EVs from both experimental groups revealing the presence of Flotillin-1, CD9 and CD81 as well as the faint signal from Albumin, co-isolated with EVs from serum.

Figure 4

Extracellular Vesicle detection by SPRi biosensor. (A): SPRi sensorgram related to the injection of 500 μL of EVs (40 μg/mL) on a chip with an array of 12 families of ligands (four spots of each family). Each curve is the average of the signal collected on the four spots of the same ligand. Each signal is the subtraction between the signal obtained on the specific ligand family and the signal obtained on the negative ctrl family (anti-IgG) spotted on the same chip. (B): SPRi intensities (average + standard error) related to the injection of EVs of 19 ischemic stroke patients and 20 CTRL subjects. The signals are normalized for the intensities collected on the anti-CD9 family spotted on each SPRi chip following a previously reported protocol [24]. ** = p < 0.05 after Mann–Whitney test.

Figure 5

Extracellular Vesicles characterization by SPRi biosensor. SPRi signals obtained on the EVs immobilized on the biosensor by secondary labeling with VEGFR2 antibody conjugated with GNPs (A) or with the TSPO ligand PK-11195 (B). The signals obtained on EVs from healthy controls (CTRL) and ischemic stroke patients (STROKE) are reported.

Figure 6

Correlation study. β regression coefficients of the Elastic-Net multivariate analysis for, respectively, the admission (A) and discharge (C) MBI models. Actual versus predicted plot with median absolute value [IQR] and R² coefficient for, respectively, the admission (B) and the discharge (D) models.