A commercial OT device measured the plasma membrane rigidity in uRBCs and synchronized iRBCs in the ring and schizont stages of P. falciparum. These measurements were based on the mean of the cell membrane spring constant (K), defined as the relation between the force applied to the cell membrane and the resulting cell elongation.

Our initial results showed that the stretching force applied to iRBCs increases proportionally to the parasite stage. Consequently, as parasites grow within RBCs, more force is needed to elongate them to the same degree as uRBCs or iRBCs in the early stages. The OT measurement showed that changes in cell elongation of uRBCs by forces as small as 15 pN could be detected. However, iRBCs in the ring and schizont stages required greater force magnitudes to reach similar deformation distances at 50 and 80 pN, respectively ( Figure 1 ).

On the other hand, the average spring constant for uRBCs was 20.65pN/μm, while iRBCs in the ring and schizont stages were 39.46 and 82.33pN/μm, respectively ( Figure 2 ). These results also showed a significant stage-dependent iRBC rigidification in the schizont stage (p =<0.0001) and in the ring stage (p = 0.0428) in comparison with uRBCs ( Table 1 ). The values for uRBCs were in accordance with previous reports using OT (Dao et al., 2003; Yoon et al., 2008).

The difference in RBC stiffness was also examined following treatment with standard drugs. We chose to use mefloquine (MQ), chloroquine (CQ), artemisinin (ART), and pyrimethamine (PYR) in a concentration four times greater than their established EC50 for 4 h ( Table 2 ). For this study, uRBCs from the same donor were evaluated with each antimalarial compound to determine whether the observed effects were specific to iRBCs. Interestingly, no antimalarial drug significantly altered the uRBCs’ rigidity ( Supplementary Figure S1 ).

It is worth noting that MQ, CQ, and ART increased the rigidity of the plasma membrane of iRBCs with schizonts. Still only MQ (p = 0.03) and ART (p< 0.0001) showed significant differences in the spring constant compared to untreated controls ( Figure 3 , Table 1 ). In contrast, PYR decreased stiffness by 7% ( Figure 3 , Table 1 ).

We also tested the effect of CQ on synchronized iRBCs in the ring stage since CQ has the most antimalarial activity in this stage (Zhang et al., 1986). In this situation, membrane stiffness was also significantly augmented in comparison with the untreated control (p = 0.03, Figure 4 ).

Our initial data showed that PYR-resistant HB3 presented a 7% decrease in stiffness of the RBC plasma membrane. Previously, it was reported that the HB3 strain is susceptible to almost all antimalarial drugs except pyrimethamine (Nzila and Mwai, 2010). Therefore, we also assessed the rigidity of the cell membrane after treatment with PYR on the 3D7 strain, a P. falciparum strain reported to be susceptible to PYR. Remarkably, the membrane rigidity of RBCs in P. falciparum 3D7 samples treated with PYR was 26% higher than that of the untreated group ( Figure 5 ).

Furthermore, we determined that changes in cell membrane rigidity depended on the strain’s sensitivity to drugs. In the case of the HB3 strain, with an experimental IC₅₀ of 68.7 μM to PYR, no significant changes were detected in cell membrane rigidity, with a median deformability of 0.93. In contrast, the PYR-sensitive 3D7 strain (IC₅₀ = 8 nM) showed a significantly increased stiffness after drug treatment with a median deformability of 1.26 and p = 0.04. Individual concentrations and stiffness values were normalized to DMSO control ( Table 3 ).

To assess the bystander effect, we first measured the stiffness of uRBCs from two independent in vitro cultures infected with HB3 and 3D7 strains, which were synchronized at the schizont stage. Then, we compared these measurements with a uRBC culture from the same donor but without the prior presence of parasites. We determined that the spring constant in uRBCs from HB3 and 3D7 cultures was 29% and 35% greater, respectively, than for those that had never been in contact with parasites ( Figure 6 , Table 4 ).

After that, we evaluated the effect of MQ, CQ, ART, and PYR in those uRBC cultures that were previously exposed to iRBCs in a standard in vitro culture. As a control, we measured the stiffness of uRBCs treated with the same drugs and cultured without P. falciparum parasites. The measurement by OT showed that the stiffness dropped by 18%, 28%, 25%, and 14% after being exposed to MQ, CQ, ART, and PYR, respectively, though only with statistical significance for CQ (p = 0.03) and ART (p = 0.0090) ( Figure 7 , Table 5 ).

The malaria parasite strains HB3 (pyrimethamine-resistant) of P. falciparum (a generous gift of the Experimental Therapeutics Division of the Walter Reed Army Institute of Research in Silver Spring, MD) and 3D7 (pyrimethamine-sensitive) (ATCC) were cultivated following the method described by Haynes et al. (1976), with some modifications. In brief, we used O+ erythrocytes in a complete medium of RPMI 1640 supplemented with 25 mM HEPES, 0.2% sodium bicarbonate, and 10% human serum. All experiments obtained blood for RBC isolation from a single donor to avoid inter-donor variability. Before blood extraction, the volunteer was required to obtain medical certificates of good health free of medication regimes. The blood was drawn with citrate phosphate dextrose adenine (CPDA) as an anticoagulant at 10% v/v after extraction. The cultures were maintained at 37°C in a gas mixture of 5% CO₂, 5% O₂, and 90% N₂ at 2% hematocrit and synchronized with alanine and a thermal cycling incubator as described in Almanza et al. (2011).

The configuration of the OT (MMI GmbH, Germany) was set on an inverted Olympus IX83 microscope with a ×100 oil immersion objective. The MBPS2 sensitive position detector and the MMI CellManipulator software were used to control the solid-state laser electronically with a wavelength of 1,070 nm at 8 W.

The optical tweezers must be calibrated each time before measuring the magnitude of the forces acting on the polystyrene particles. Optical stiffness, K, was determined by the oscillatory drag force method. For further measurements, forces were calculated for the optical tweezers using Hooke’s law:

where K is the optical stiffness of the trap, and x is the displacement from the trap’s center ( Supplementary Figures S2, S3 ).

The K constant of each cell was calculated by data analysis through image processing using ImageJ software. The boundary of each cell was detected in each frame for the same group of images. The changes in cell length were used along with the restoring force and Hooke’s equation to calculate the K constant ( Supplementary Figure S4 ).

RBC samples were prepared by taking previously processed RBCs. The RBCs were washed twice with PBS and re-suspended in PBS and ACD (citric acid, sodium citrate, and dextrose). Subsequently, polystyrene particles with a diameter of 3 μm, previously washed with PBS and left at 4°C for 1 h to obtain a better adherence of the particles to the RBC, were added. Then, polystyrene particles were used to deform the selected uRBC or iRBC. OT was used to manipulate the cell only when one bead was attached to the membrane’s edge, and the cell’s opposite side was attached to the glass plate, allowing stretching without shedding. The experiments were performed in the linear regime, as all deformation vs. force curves present this behavior. The cell also always recovered its initial shape after each stretching ( Supplementary Figure S5 ).

Magnetic columns were used to enrich the population of late-stage parasites following the methodology of Coronado et al. (2013). The isolated parasites were washed twice with PBS and diluted in PBS and ACD. Polystyrene particles were added and left at 4°C for 1 h. Each sample was then visualized under the microscope, and OT stretched the cell.

In the case of the drug-treated group, the samples were incubated previously with concentrations of chloroquine (CQ) 1 μM, mefloquine (MQ) 1 μM, artemisinin (ART) 20 nM, and pyrimethamine at 20 nM for 4 h as described previously by Deng et al. (2015) ( Supplementary Figure S6 ). The samples were incubated at 37°C. Posteriorly, polystyrene particles were added and incubated at 4°C for 1 h. The samples were then placed under the microscope, and the cell was stretched. We also evaluated the bystander effect with the drugs mentioned above using the same conditions and incubation time. For the bystander effect evaluation, uRBCs were in contact with parasites for 48 h prior to experimentation, and all assays were performed while the parasites in culture were in schizont stage.

Relative deformability, RD, expresses the deformability of the treated cells, with D_t normalized to the median deformability of cells in the DMSO control for a given experimental series, D _ctrl, according to the following formula:

This procedure facilitates the assessment of the effects of treatment on deformability and is easily compared between experiments.

All experiments were independently repeated at least twice, and 15–20 cells were analyzed per sample. The dispersion graphs of all experiments are presented. Statistical analysis was calculated using Prism Gradpad v7 with a t-test when comparing one group to another. Multiple comparison analysis was performed with one-way ANOVA. Data were evaluated by analysis of variance, and significant differences between groups were determined using Bonferroni’s modification. The relationship between the force applied and the changes in RBC diameter was evaluated by linear regression to calculate the K spring constant. P< 0.05 was considered statistically significant. Significance was considered as follows: *p< 0.05; **p< 0.01; ***p< 0.005, ****p< 0.0001.