Contact-free single cell assay for assessing RBC mechanical properties using brillouin microscopy: Evaluation of anti-sickling therapeutics

Abstract Background: Brillouin microscopy is a non-contact, all-optical, label free technique, recently developed for biomechanical studies (elastic modulus and viscoelasticity). It is based on spontaneous Brillouin light scattering, an interaction between incident light and thermally generated acoustic waves (phonons) within a sample resulting in Doppler-like frequency shift in the scattered light in GHz range, corresponding to the propagation of mechanical (acoustic) waves. The magnitude of this frequency shift is directly proportional to the longitudinal elastic modulus, providing insights into RBC deformability. Although various biological tissues have been studied with Brillouin microscopy, this is the first application for single-RBC imaging. Sickle cell disease (SCD) phenotype is notable for impaired RBC deformability, current methods to measure deformability require application of shear stress, which itself alters deformability; Brillouin imaging offers ‘touchless’ quantification of RBC elastic modulus. 5-Hydroxymethylfurfural (5-HMF) is an antisickling-drug which binds to the N-terminal of β-globin chain of HbS forming a high-affinity Schiff-base adduct, which increases Hb-O2 affinity thus preventing RBC sickling, thereby increasing the cell deformability. In the current study, we developed a touchless single-cell RBC imaging method to analyze the effect of 5-HMF upon SCD-RBC deformability. Method: Sickle RBCs were fractionated from SCD patient's blood, using percoll-based density gradient centrifugation and were incubated with 2.5 and 5 mM 5-HMF for 1 hour/37°C. The single RBCs (n=19-31) were imaged using Brillouin microscopy under varying O2 conditions using controlled environmental chamber (oxygenation-21%O2, deoxygenation-1%O2and reoxygenation-95%O2) to assess the Brillouin frequency shift during deoxygenation/reoxygenation cycling. The Brillouin microscopy system utilized a 660 nm continuous-wave laser (Torus, Laser Quantum Inc.) that was focused using a high-numerical-aperture objective lens (Olympus 40x / 0.95 NA) mounted on an inverted microscope (Olympus IX81). The Brillouin imaging was performed using a motorized XY stage with a step size of 0.5 µm for spatial scanning. We also performed the oxygen scan of intact sickle RBCs (using LORRCA MaxSis RBC Analyzer) to assess the point of sickling and point of recovery. Statistical analyses were done using GraphPad Prism software 10.5. Results: There was a significant decrease in the Brillouin frequency shift (corresponds to increased deformability) of SCD-RBCs at 2.5 mM and 5 mM 5-HMF doses under oxygenated (6.25±0.07 vs. 6.16±0.10 vs. 6.16±0.05 GHz; 0 vs. 2.5 vs. 5 mM 5-HMF; p<0.001*) and reoxygenated conditions (6.21±0.05 vs. 6.17±0.06 vs. 6.16±0.07 GHz, 0 vs. 2.5 vs. 5 mM 5-HMF; p<0.01*). The Brillouin frequency shift was significantly reduced in 2.5 mM treatment group (6.27±0.08 vs. 6.21±0.09, 0 vs. 2.5 mM 5-HMF; p<0.05) in deoxygenated condition. Similarly, we observed a significant decrease in point of sickling (77.09±9.68 vs. 56.82±18.65 vs. 37.58±17.13- 0 vs. 2.5 vs. 5 mM 5-HMF; p<0.0001*), and in point of recovery (76.4±18.38 vs. 50.69±19.68 vs. 37.93±12.75- 0 vs. 2.5 vs. 5 mM 5-HMF; p<0.0001*). Conclusion: This study demonstrates that Brillouin microscopy is a powerful technique which can be used for touchless single RBC imaging to assess biomechanics of single cell during physiologic deoxygenation/reoxygenation using a controlled environmental chamber. 5-HMF improved sickle RBCs biomechanics resulting in increased deformability as evidenced by decreased Brillouin frequency shift. The optimum deformability was observed at 2.5 mM 5-HMF dose across oxygenated, deoxygenated and reoxygenated conditions. Future directions: We will be assessing the anti-sickling effect of in-house reformulated, RBC-targeted 5-HMF prodrugs [Lipid prodrug (LP), Carbon Dot (CD), Compound Nanoparticle (CompNP)] using Brillouin microscopy.