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Biomimetic proteolipid vesicles for reverting GPI deficiency in paroxysmal nocturnal hemoglobinuria

Biomimetic proteolipid vesicles for reverting GPI deficiency in paroxysmal nocturnal hemoglobinuria

Authors: Valentina Giudice, Pasqualina Scala, Erwin P. Lamparelli, Marisa Gorrese, Bianca Serio, Angela Bertolini, Francesca Picone, Giovanna Della Porta, and Carmine Selleri


Nano-vesicular carriers are promising tissue-specific drug delivery platforms. Here, biomimetic proteolipid vesicles (BPLVs) were used for delivery of glycosylphosphatidylinositol (GPI)-anchored proteins to GPI deficient paroxysmal nocturnal hemoglobinuria (PNH) cells. BPLVs were assembled as single unilamellar monodispersed (polydispersity index, 0.1) negatively charged (ζ-potential, −28.6 ± 5.6 mV) system using microfluidic technique equipped with Y-shaped chip. GPI-anchored and not-GPI proteins on BPLV surface were detected by flow cytometry. Peripheral blood mononuclear cells (PBMCs) from healthy and PNH subjects were treated with BPLVs (final concentration, 0.5 mg/mL), and cells displayed an excellent protein uptake, documented by flow cytometry immunophenotyping and confocal microscopy. BPLV-treated cells stressed with complement components showed an increased resistance to complement-mediated lysis, both healthy and PNH PBMCs. In conclusion, BPLVs could be effective nanocarriers for protein transfer to targeted cells to revert protein deficiency, like in PNH disease. However, further in vivo studies are required to validate our preclinical in vitro results.

Fig 1. Cytotoxicity on healthy and paroxysmal nocturnal hemoglobinuria (PNH) peripheral blood mononuclear cells (PBMCs). (A) Cells were treated with increasing concentrations of empty (without human membrane proteins) or loaded (with proteins) biomimetic proteolipid vesicles (BPLVs) for 24 h, and cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide (MTT) assay. A sample without BPLV treatment was used as negative control (CTR-), while a sample treated with dimethyl sulfoxide (DMSO) as positive control (CTR+). (B) Cell viability was tested by MTT assay at 24 h, 48 h, and 72 h using a final concentration of 0.5 mg/mL of BPLVs. Data are represented as mean G SD. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Fig 2. Biomimetic proteolipid vesicle (BPLV) uptake by healthy peripheral blood mononuclear cells (PBMCs). Vesicle uptake was studied by confocal microscopy, as BPLVs were tagged with PE-rhodamine tracer (red signals), while cell nuclei with 40,6-diamidino-2-phenylindole (DAPI; blue signals), and GPI-anchored proteins with Flaer-Alexa Fluor 488 (green signals). PBMCs were treated (w) or not (w/o) with BPLVs for 48 h and 72 h, and a baseline (T0) sample was used as control. PBMCs that have uptake BPLVs showed an intense merged purple signal, showing colocalization of PE-rhodamine vesicles and GPI-anchored proteins carried by BPLVs.

Selected Figures

Keywords: Drug delivery system; natural sciences; biological sciences; biochemistry; biomolecules; lipid nanoparticles; Flex-M

iScience 2024, 27 (3), 109021


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