A Silicon Rhodamine-fused Glibenclamide to Label and Detect Malaria-infected Red Blood Cells

Abstract

The malaria parasite Plasmodium falciparum affects the lives of millions of people worldwide every year. The detection of replicating parasites within human red blood cells is of paramount importance, requiring appropriate diagnostic tools. Herein, we design and apply a silicon rhodamine-fused glibenclamide (SiR-glib). We first test this far-red fluorescent, fluorogenic and endoplasmic reticulum-targeting sulfonylurea in mammalian cells and pancreatic islets, before characterizing its labeling performance in red blood cells infected with the asexual developmental stages of Plasmodium falciparum. We further combine SiR-glib with a portable smartphone-based microscope to easily and rapidly identify parasitized red blood cells, providing proof of principle for diagnostic use in rural endemic areas without major healthcare facilities.

Keywords: Malaria; Plasmodium falciparum; Red Blood Cells; Silicon Rhodamine; Smartphone-Based Detection.

Figure 1

Silicon rhodamine‐fused glibenclamide for the detection of P. falciparum infected red blood cells (RBCs). A) RBC do not contain any organelles, which changes through the infection by P. falciparum parasites due to host cell remodeling and genesis of ER, nucleus, etc in the parasite. B) High absorbance of oxygen free (Hb) and oxygen‐bound hemoglobin (HbO₂) in the UV to green range might mask ER tracker green. Far‐red silicon rhodamine falls into the biological imaging window above 600 nm. C) Chemical structure of glibenclamide and its fluorophore‐linked congeners ER tracker green (BODIPY fused) and SiR‐glib (SiR fused).

Scheme 1

Synthesis of SiR‐glib. Commercially available benzoic acid 1 is peptide coupled after TSTU activation to obtain 2, on which the sulfonylurea is installed using cyclohexyl isocyanate. Zn‐mediated reduction of the nitro group yields aniline 4, on which an alkyl amine linker is installed using an in situ formed acyl chloride before subsequent Fmoc‐deprotection. Silicon rhodamine is finally fused onto 6 by using its NHS‐activated ester, yielding SiR‐glib.

Figure 2

Characterization of SiR‐glib. A) SiR‐glib may adopt two different isomeric forms, a non‐fluorescent spirolactone and an open, fluorescent zwitterionic form. B) Excitation and emission spectra of SiR‐glib, showcasing its fluorogenicity by addition of SDS. C) Fluorescence pH dependency of SiR‐glib. mean±SD. n=3. D) Live cell imaging by confocal microscopy using ER tracker green and SiR‐glib in live HeLa cells and live primary astrocytes. E) Live cell imaging by confocal microscopy of the beta‐cell marker LUXendin551 and SiR‐glib in live islets of Langerhans. SiR‐glib gives rise to cytoplasmic and membrane structures, presumed to be ER‐retained SUR1 and K_ATP channels, respectively. Scale bars=15 μm.

Figure 3

Labeling of two different P. falciparum strains with SiR‐glib. A) The P. falciparum strain 3D7 was grown in a 5 % HCT culture. Cells were labeled with 2 μM SiR‐glib, 2 μM SiR or remained untreated for 1 h. Only SiR‐glib‐treated cells showed a specific labeling of the ring (R), trophozoite (T) and schizont (S) developmental stages of the parasite for both investigated strains. Signals of all stages are included (All). Scale bars=3 μm. B) As for (A) but using the P. falciparum strain FCR3. C, D) Quantification of fluorescence from (A) and (B) reveals a significantly stronger fluorescence of SiR‐glib‐treated RBCs compared to using SiR or untreated RBCs in every developmental stage. N=3; n≥11. *: p <0.05; **: p <0.01; ***: p <0.001. E, F) As for (C) and (D) but in a 40 % HCT culture shows lower signal in ring stages when compared to 5 % HCT. N=3; n≥12. *: p <0.05; **: p <0.01; ***: p <0.001. t‐Test or Mann‐Whitney Rank Sum test.

Figure 4

Imaging and analysis of P. falciparum infected RBCs using a portable smartphone‐based microscope. A) Sample preparation for smartphone‐based imaging. B) Workflow showing the mounted smartphone, image recording and background subtraction (photo reproduced from ref. and licensed under CC BY 4.0). C) Vehicle controls of uninfected and 3D7‐ or FCR3‐infected RBCs. D) Analysis of vehicle‐treated RBCs show a significant increase in pixels above threshold for FCR3‐infected RBCs. mean±SD. n=6. ***: p <0.005; ns=non‐significant. Ordinary one‐way ANOVA. E) As for (C) but SiR‐glib‐treated RBCs. F) Analysis of SiR‐glib‐treated RBCs show a significant increase in pixels above threshold for RBCs infected with both strains, also enabling the detection of the 3D7 strain. mean±SD. n=6. ****: p <0.001. Ordinary one‐way ANOVA.