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. 2023 Jan 16;21(1):15.
doi: 10.1186/s12951-022-01709-x.

Erythrocyte membrane with CLIPPKF as biomimetic nanodecoy traps merozoites and attaches to infected red blood cells to prevent Plasmodium infection

Zhouqing He  1 , Chuyi Yu  1 , Ziyi Pan  1 , Xiaobo Li  1 , Xiangxiang Zhang  1 , Qijing Huang  1 , Xingcheng Liao  1 , Jiaoting Hu  1 , Feng Zeng  1 , Li Ru  1 , Wanlin Yu  2 , Qin Xu  1 , Jianping Song  1 , Jianming Liang  3   4
Affiliations

Erythrocyte membrane with CLIPPKF as biomimetic nanodecoy traps merozoites and attaches to infected red blood cells to prevent Plasmodium infection

Zhouqing He et al. J Nanobiotechnology. .

Abstract

Background: Malaria remains a serious threat to global public health. With poor efficacies of vaccines and the emergence of drug resistance, novel strategies to control malaria are urgently needed.

Results: We developed erythrocyte membrane-camouflaged nanoparticles loaded with artemether based on the growth characteristics of Plasmodium. The nanoparticles could capture the merozoites to inhibit them from repeatedly infecting normal erythrocytes, owing to the interactions between merozoites and heparin-like molecules on the erythrocyte membrane. Modification with a phosphatidylserine-targeting peptide (CLIPPKF) improved the drug accumulation in infected red blood cells (iRBCs) from the externalized phosphatidylserine induced by Plasmodium infection. In Plasmodium berghei ANKA strain (pbANKA)-infected C57BL/6 mice, the nanoparticles significantly attenuated Plasmodium-induced inflammation, apoptosis, and anemia. We observed reduced weight variation and prolonged survival time in pbANKA-challenged mice, and the nanoparticles showed good biocompatibility and negligible cytotoxicity.

Conclusion: Erythrocyte membrane-camouflaged nanoparticles loaded with artemether were shown to provide safe and effective protection against Plasmodium infection.

Keywords: Artemether; Erythrocyte membrane biomimetic nanomaterials; Merozoites; Targeted delivery; pbANKA-infected malaria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Schematic illustration of PEM-ARM. PEM-ARM was fabricated by surrounding the erythrocyte membrane with artemether liposomes modified with the CLIPPKF peptide. The two components of PEM-ARM made it capable of simultaneously targeting merozoites and infected erythrocytes in pbANKA-infected mice
Fig. 2
Fig. 2
Preparation and characterization of PEM-ARM. A Schematic diagram of the preparation process of PEM-ARM. B Protein composition analyses of RBCm, Lip-ARM and PEM-ARM using SDS-PAGE. C Representative TEM images of Lip-ARM, EM-ARM, and PEM-ARM; erythrocyte membrane (blue arrow), scale bar: 100 nm. D Mean diameter and zeta potential of Lip-ARM, EM-ARM, and PEM-ARM. Data points represent mean ± s.d. (n = 3). E Stability of liposomes in water (n = 3). F Relative particle size and G relative turbidity in 10% serum of liposomes (n = 3)
Fig. 3
Fig. 3
iRBCs-binding capacity in mice with malaria. A Analysis of the growth cycle of erythrocytes in pbANKA-infected mice through the HO/TO double-staining method as assessed by flow cytometry. B Normal erythrocytes, C rings, D trophozoites, and E schizonts took up liposomes in mice with different infection rates (n = 3, * P < 0.05 and ** P < 0.01). F Confocal fluorescent images of iRBCs (blue), PEM-ARM (red), and the everted PS on the surface of iRBCs (yellow) are shown. Normal erythrocyte (white arrow), scale bars: 20 μm in general; 2 μm in normal erythrocyte, trophozoite, and schizont
Fig. 4
Fig. 4
Neutralization of merozoites. A Purification of mature iRBCs with Percoll separation solution, illustrating iRBCs (red arrow) and normal erythrocytes (blue arrow). B, C Uptake of Lip-ARM, EM-ARM, and PEM-ARM by merozoites in pbANKA-infected mice (n = 3, * P < 0.05 and ** P < 0.01). D Confocal fluorescent images of merozoites (blue) and PEM-ARM (red); scale bar = 20 μm (general), scale bar = 2 μm (magnification). E Representative scatter plots of Hoechst 33,342/CFDA-SE analysis of the cytoadherence assay of merozoites and normal erythrocytes after drug treatment. F Percentage of Q2 area cells (n = 3, * P < 0.05 and ** P < 0.01)
Fig. 5
Fig. 5
Therapeutic effect of PEM-ARM in vivo. A Sketch of the therapy regimen. B Survival curve of C57BL/6 mice exposed to pbANKA infection. C Weight change curves of C57BL/6 mice. D Flow cytometry detection of infection rate in mice (n = 5, * P < 0.05 and ** P < 0.01). E Ring, F trophozoite, and G schizont stages of iRBCs changed in proportion. (n = 5, * P < 0.05 and ** P < 0.01). H Reactive oxygen species and I mitochondria activity analysis of iRBCs treated with different drugs (n = 3, * P < 0.05 and ** P < 0.01). J Mice lungs infected with pbANKA or treated with PBS, f-ARM, Lip-ARM, EM-ARM, or PEM-ARM, with mock-infected mice lungs as a control
Fig. 6
Fig. 6
Characterizations of several measures of the safety of PEM-ARM. A Fluorescence images and B Median fluorescence intensity charts of MLE-12 cellular uptake after liposome treatment. Scale bar = 200 μm. Cell nuclei and the liposomes were labeled with HO (blue) and NR (red), respectively (n = 3, * P < 0.05 and ** P < 0.01). C MTT assay after MLE-12 cells were incubated with various concentrations of liposomes (n = 6, * P < 0.05 and ** P < 0.01). D H&E-stained slices of the liver, spleen, lung, kidney, brain, and heart. Scale bar = 200 μm, hemozoin (red arrow), glomerular capillary congestion sign (black frame)
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