Mutual interaction of red blood cells influenced by nanoparticles

Despite extensive studies on different types of nanoparticles as potential drug carriers, the application of red blood cells (RBCs) as natural transport agents for systemic drug delivery is considered a new paradigm in modern medicine and possesses great potential. There is a lack of studies on the influence of drug carriers of different compositions on RBCs, especially regarding their potential impact on human health. Here, we apply conventional microscopy to observe the formation of RBC aggregates and optical tweezers to quantitatively assess the mutual interaction of RBCs incubated with inorganic and polymeric nanoparticles. Scanning electron microscopy is utilized for direct observation of nanoparticle localization on RBC membranes. The experiments are performed in a platelet-free blood plasma mimicking the RBC natural environment. We show that nanodiamonds influence mutual RBC interactions more antagonistically than other nanoparticles, resulting in higher aggregation forces and the formation of larger cell aggregates. In contrast, polymeric particles do not cause anomalous RBC aggregation. The results emphasize the application of optical tweezers for the direct quantitative assessment of the mutual interaction of RBCs influenced by nanomaterials.


Optical tweezers experimental approach
A double-channel optical tweezers (OT) experimental system (Fig.S1) is used for RBC manipulation at a single-cell level by two focused laser beams [1,2]. A beam from single-mode Nd:YAG infrared laser ( = 1064 nm, max power 350 mW, ILML3IF-300 Leadlight Technology, Taiwan) is expanded by lenses L1 and L2 and then is divided into two separate beams by beam splitter BS1 to control the overall incident beam power. λ/2 waveplates and beam splitter BS2 are used for regulation of power ratio between two trapping beams. The position of the moveable trap inside the sample is controlled in the focal plane of the focusing objective by a conjugated beam adjustable mirror (M2). A water immersion objective LUMPlanFl 100× (Olympus, Japan) with a large numerical aperture (NA=1.00) allows focusing the two laser beams and forming optical traps inside the sample chamber. The sample chamber is based on the microscopy slide and cover glass separated by double-side adhesive tape (~ 100 μm thick). In the transmission mode the samples are illuminated with white LED from the bottom of sample chamber (see Fig.S1), and the trapped RBC are visualized by CMOS camera (Pixelink PL-B621M, Canada). To measure the force applied to RBC during cell trapping, calibration procedure is required. It is done by finding the maximal returning force through the matching of the trapping force with the external (viscous friction) force Fdrag exerted on the RBC. The force of the viscous friction is calculated by the Stokes' law (defined by the force exerted on a particle in a viscous fluid): where the dynamic viscosity of blood plasma at room temperature 25°C is η = 1.66×10 −3 Pas [1], r is the RBC radius considering RBC to be equivolume spheres with the radius r = 2.7 μm, ν is the flow velocity relative to the object, K is the correction factor defined empirically [2]. The 10-15 RBC are used for the calibration procedure for each laser beam at different laser power values. The calibration is done for RBC incubated with NP but no difference is observed compared to untreated RBC.
RBC with the large NP aggregates (~ 1 μm) on the cell surface can be trapped with OT but the manipulation of the cell is difficult to implement in this case. The RBC-NP complex is hard to lift for interaction force measurements, arguably because the NP aggregates drastically increase the weight of RBC or change overall geometry of RBC-NP complex making the trap less efficient. Whereas, small NP aggregates (less than ~ 1 μm) localised on the RBC membrane do not influence the manipulation and/or aggregation forces in comparison to RBC samples without NP.

Aggregation force measurement
To quantify the RBC cell-to-cell interaction, the aggregation force Fa, the minimum force required to stop RBC from overlapping [3,4] is measured.
Two RBC trapped by two laser beams (beam 1 and beam 2, see Fig.S2; power of each beam does not exceed 50 mW) are moved in contact to each other to form an aggregate with ta small interaction area (~ 3 µm in diameter); the process takes about 10 s. Then, the power of one laser beam is slowly decreased until the RBC start to overlap spontaneously by forming a doublet. We assume that the optical trapping force F (corresponding to the power of the beam losing the RBC as a result of this procedure) is equal to the interaction force between the RBC in the aggregate.

Interaction energy calculation
The interaction energy is assessed quantitatively based on the measured interaction force as described in [4,5]. The results are presented as a function of interaction energy per conjugated surface area of interacting RBC vs. the interaction area related to the area of a single red blood cell (S/SRBC).