Nanoparticle tracking for the detection of quantitative changes in circulating microvesicles following remote ischemic preconditiong (RIPC)

Objective: A pivotal role in the systemic intercellular cross-talk is suggested for circulating microvesicles (cMV) and a variety of pathologies and treatment regimens are associated with a quantitative and qualitative alternation of cMV levels. The aim of this investigation was to implement nanoparticle tracking (NPT) for the verification of current cMV isolation protocols following repetitive transient limb ischemia, as applied for the induction of remote ischemic preconditioning (RIPC).

Methods: Blood samples of five healthy volunteers were taken immediately before and 20 min after 3 × 5 minutes of blood pressure cuff inflation with 200 mmHg on the left upper arm. cMV isolation was performed after 3 steps of centrifugation with 1500 g for 5 minutes, 2800 g for 20 minutes and 100000 g for 90 minutes. Supernatant and pellet were processed separately for the quantitative detection of cMV by NPT.

Results: NPT was able to detect cMV with a size span of ca. 60nm up to several hundred nm, thus including both, the exosome and microparticle fractions. In compliance with previous reports using flow cytometry, NPT demonstrated a large biological inter-individual variation of cMV. After differenzial centrifugation, cMV were detected in the pellet (range concentration 7.433 – 121.2 particles/mlx10⁶), but were surprisingly also present in the supernatant (concentration 0.900 – 31.900 particles/mlx10⁶). Application of RIPC led to a decrease in the overall concentration of cMV after 20 minutes (59.13 ± 43.29 vs. 41.02 ± 37.69 particles/mlx10⁶ p < 0.05), with no significant difference between the supernatant fractions (13.17 ± 11.89 vs. 5.91 ± 2.89 particles/mlx10⁶ p = 0.094). When NPT was applied to determine the size distribution, no significant differences between the analysed individuals (diameter of 50% of MVs (X50) range: 0.036 – 0.146 µm) was detected. Moreover, RIPC had no impact on the size of detected cMV (pellet X50: 0.095 ± 0.0371 µm vs. 0.102 ± 0.0324 µm p = 0.096) (supernatant X50: 0.128 ± 0.013 µm vs. 0.138 ± 0.009 µm p = 0.065).

Conclusion: As a powerful, high sensitive tool for the detection of small and large size cMV, NPT represents an ideal addition to established methods, such as flow cytometry. NPT reveals that RIPC may have an impact on the number of cMV in healthy volunteers without an effect on their size distribution. The use of NPT may help to further standardize and improve current cMV isolation protocols.