Microwaves can kill malaria parasites non-thermally

Abstract

Malaria, which infected more than 240 million people and killed around six hundred thousand only in 2021, has reclaimed territory after the SARS-CoV-2 pandemic. Together with parasite resistance and a not-yet-optimal vaccine, the need for new approaches has become critical. While earlier, limited, studies have suggested that malaria parasites are affected by electromagnetic energy, the outcomes of this affectation vary and there has not been a study that looks into the mechanism of action behind these responses. In this study, through development and implementation of custom applicators for in vitro experimentation, conditions were generated in which microwave energy (MW) killed more than 90% of the parasites, not by a thermal effect but via a MW energy-induced programmed cell death that does not seem to affect mammalian cell lines. Transmission electron microscopy points to the involvement of the haemozoin-containing food vacuole, which becomes destroyed; while several other experimental approaches demonstrate the involvement of calcium signaling pathways in the resulting effects of exposure to MW. Furthermore, parasites were protected from the effects of MW by calcium channel blockers calmodulin and phosphoinositol. The findings presented here offer a molecular insight into the elusive interactions of oscillating electromagnetic fields with P. falciparum, prove that they are not related to temperature, and present an alternative technology to combat this devastating disease.

Keywords: Plasmodium falciparum; Radio frequencies (RF); calcium signaling; electromagnetic fields; electromagnetism; malaria; microwaves (MW); therapeutic.

Figure 1

Microwave exposure system and their components. (A) Block diagram of all parts integrating the G and M3 microwave applicator systems (experiments were conducted separately); (B) M3 and (C) WG applicator device drawings. (D) Placement of the sample positioned under the metallic strip on a glass slide with a laser-milled cavity and the thermometer probe in the M3 (E) Test tube that holds inside the microtube that will carry the sample during microwave exposure inside the WG; the number 1 shows the microfuge (F) Drawing of the sample holders for the WG with the three thermometer probes placed at different depths of the sample. Dimensions are not to scale.

Figure 2

FEM SAR modeling and validation. Electromagnetic model of (A) WG and (B) M3; applicators showing calculated maximum local SAR intensity distribution in the sample. To the right of each model are the measured and simulated relative reflected power over frequency (scattering parameter S11).

Figure 3

Non thermal effects of MW on P. falciparum parasites or mammalian cells. (A) Representative pattern of temperature changes caused by exposure of P. falciparum-infected cultures to MW. Infected cultures, epithelial (Vero) cells or J-774 macrophages were untreated or exposed to MW in the (A, B) WG or (C) M3 and the growth or viability of duplicates was assessed 24 h after treatment. Data was acquired through: A, flow cytometry for HB3 (n=3), and fluorometry with sulforhodamine for cells (n=2); B, MTT assays for all; (HB3, n=1; Vero cells and macrophages, n=3); C, flow cytometry for HB3 (n=3) and MTT assays for cells treated with M3 (n=3). (D) Fluctuations of temperature of the infected, exposed culture with both MW applicators. (E) Using a representative temperature profile generated in the WG, samples were either submitted to the same temperature fluctuations by using a thermocycler, or exposed to the MW treatment in the WG, and their growth was monitored 24 h later by flow cytometry. A representative experiment is shown. n=3. One-way ANOVA was used throughout. (F) Growth of thermo- or MW- treated parasites was monitored for 72 hours by flow cytometry. One representative experiment is shown. n=2. **p<0.01, ***p<0.005.

Figure 4

Ultrastructural changes in iRBCs after treatment with MW. MW-treated or untreated parasites were examined by transmission electron microscopy up to 12 h after MW treatment. The scale bar for Control 12 h is the same as MW sample 12 h. PM, Parasitophorous membrane; RBC, red blood cell; FV, food vacuole; HZ, haemozoin; N, nucleus; M, merozoite; Rh, rhoptry; GC, Golgi complex.

Figure 5

Calcium and pH changes in MW-exposed infected RBCs. (A) Localization of Ca²⁺ with time progression under microscopy of iRBCs stained with the calcium dye, seen under fluorescence (second and fourth columns) or bright light (first and third columns) corresponding to the same time points. with no treatment or MW exposure (I.A, immediately after end of exposure). The pictures are representative images of one experiment out of two. (B) Measurement of cytosolic Ca²⁺ with the use of a fluorometer. The graph has been modified to show the time elapsed during treatment, in which no measurement was taken. A first reading was taken before exposure; the next ones, after the end of exposure, repeating the measurements every 10 min. A representative result, out of two, is shown. (C) The negative change in pH of samples after MW treatment was compared to untreated samples. Chloroquine and NH₄Cl treatment were used as positive controls for acidification. n=3. Bars represent the mean ± SEM values; one-way ANOVA was used.

Figure 6

Molecular damage triggers apoptosis in MW exposed parasites. (A) ROS production of parasitized or uRBCs was assessed. Hydrogen peroxide was used as a positive control at a final concentration of 200 μM. ROS production in Vero cells or in macrophages was analyzed after exposure to the same treatment as that of RBCs or iRBCs. Bars show the mean intensity fluorescence of triplicates, of three experiments read in a fluorometer. (B) Lipid peroxidation was detected through a Click-It LAA reaction. Cumene hydroperoxide at 100 uM was used as a positive control. Bars show the relative fluorescence units of triplicates of one representative experiment out of three read in a fluorometer. (C) Caspase-like activity measured through flow cytometry. Data are expressed as % of control and are the means of duplicates, n=3. (D) DNA fragmentation was quantified by flow cytometry 24 hours after MW treatment. Experiments were run in triplicates, n=3. Data are the means ± SEM. (E) Level of phosphatidylserine externalization after MW irradiation on uRBC (left) or iRBCs (right) using an Annexin V assay to measure its level. Staurosporine was used as positive controls for phosphatidylserine externalization. Data are the means of n=3 ± SEM. (F) The mitochondrial membrane potential was measured through flow cytometry measurement of red JC-1 fluorescence which correlates with the mitochondrial membrane potential ΔΨ_m. Bar graphs show the geometric mean of FL2 (red fluorescence) using 50 µM CCCP as positive control for mitochondrial membrane depolarization. Data are the mean of n=3 ± SEM. Two-way ANOVA analysis was used in (A) and (B) and one-way ANOVA was used in (C-F). *p<0.05, **p<0.01, ***p<0.005.

Figure 7

Assessment of necrosis or autophagy induced by MW exposure. (A) Levels of PI intake of uRBCs or iRBCs after MW treatment. Bars represent the mean relative fluorescence intensities of two independent experiments ± SEM values read in a fluorometer. One-way ANOVA analysis was used.(B) iRBCs were incubated with or without an autophagy inhibitor (3-MA) before exposing them to MW, or placed in starvation media as a positive control; n=3 ± SEM values; two-way ANOVA was used. Readings were obtained through flow cytometry. (C) Transmission electron microscopy (TEM) image of P. falciparum infected red blood cells six hours post microwave treatment. Arrows point to doubled-membrane autophagosomes. (D) Flow cytometry analysis of infected erythrocytes exposed to MW shows changes in size (FSC) and complexity (SSC). Data are the means of n=3 ± SEM. T-test analysis was used. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Figure 8

Ca²⁺ signaling proteins involved in MW-induced death. uRBCs or iRBCs were incubated 1 h before MW in WG with or without inhibitors of Ca²⁺ transduction pathways, and their growth assessed by flow cytometry (Bars from left to right: Untreated, bromophenyl bromide, TMB8, neomicin, W7, verapamil, and indomethacine). All bars represent means ± SEM of duplicates. n=2, unpaired, one-way ANOVA (Bonferroni’s multiple comparison). *p<0.05,**p<0.01, ***p<0.005.

Figure 9

Effect of MW on the crystallization of haemozoin. A haemozoin formation assay based on the differential solubilities of haem and haemozoin was conducted in the presence of different agents by exposing infected erythrocytes to chloroquine, mefloquine or MW. (A) FT IR profile of haem from the samples and (B) FT IR profile of haemozoin. Arrows point to the characteristic peaks of both molecules. (C) Comparison of the effect of different treatments in the production of haemozoin. The values are the means of triplicates of one representative experiment (n=3). One-way ANOVA analysis was used. ***p<0.005.

Figure 10

Proposed pathways involved in MW-induced death of malaria parasites. Movements of Ca²⁺ from this ion storages of the parasite take place after MW exposure. Their inhibition with target molecules can partially or totally prevent their death by irradiation. (PVM: Parasitophorous vacuole membrane, ER: Endoplasmic reticulum, FV: Food vacuole, PGE2: Prostaglandin E2, PLA: Phospholipase A, IP3: Inositol Trisphosphate, CAM: Calmodulin).