title: Immunoliposome-mediated drug delivery to Plasmodium-infected and non-infected red blood cells as a dual therapeutic/prophylactic antimalarial strategy.
abstract: One of the most important factors behind resistance evolution in malaria is the failure to deliver sufficiently high amounts of drugs to early stages of Plasmodium-infected red blood cells (pRBCs). Despite having been considered for decades as a promising approach, the delivery of antimalarials encapsulated in immunoliposomes targeted to pRBCs has not progressed towards clinical applications, whereas in vitro assays rarely reach drug efficacy improvements above 10-fold. Here we show that encapsulation efficiencies reaching >96% are achieved for the weak basic drugs chloroquine (CQ) and primaquine using the pH gradient loading method in liposomes containing neutral saturated phospholipids. Targeting antibodies are best conjugated through their primary amino groups, adjusting chemical crosslinker concentration to retain significant antigen recognition. Antigens from non-parasitized RBCs have also been considered as targets for the delivery to the cell of drugs not affecting the erythrocytic metabolism. Using this strategy, we have achieved unprecedented complete nanocarrier targeting to early intraerythrocytic stages of the malaria parasite for which there is a lack of specific extracellular molecular tags. Immunoliposomes studded with monoclonal antibodies raised against the erythrocyte surface protein glycophorin A were capable of targeting 100% RBCs and pRBCs at the low concentration of 0.5μM total lipid in the culture, with >95% of added liposomes retained on cell surfaces. When exposed for only 15min to Plasmodium falciparum in vitro cultures of early stages, free CQ had no significant effect on the viability of the parasite up to 200nM, whereas immunoliposomal 50nM CQ completely arrested its growth. In vivo assays in mice showed that immunoliposomes cleared the pathogen below detectable levels at a CQ dose of 0.5mg/kg, whereas free CQ administered at 1.75mg/kg was, at most, 40-fold less efficient. Our data suggest that this significant improvement is in part due to a prophylactic effect of CQ found by the pathogen in its host cell right at the very moment of invasion.
authors.affiliation: Nanomalaria Group, Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 10-12, ES-08028 Barcelona, Spain; Barcelona Institute for Global Health (ISGlobal, Hospital Clínic-Universitat de Barcelona), Rosselló 149-153, ES-08036 Barcelona, Spain; Nanoscience and Nanotechnology Institute (IN2UB), University of Barcelona, Martí i Franquès 1, ES-08028 Barcelona, Spain.
authors.forename: Ernest
authors.initials: E
authors.lastname: Moles
authors.affiliation: Nanomalaria Group, Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 10-12, ES-08028 Barcelona, Spain; Barcelona Institute for Global Health (ISGlobal, Hospital Clínic-Universitat de Barcelona), Rosselló 149-153, ES-08036 Barcelona, Spain; Nanoscience and Nanotechnology Institute (IN2UB), University of Barcelona, Martí i Franquès 1, ES-08028 Barcelona, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Urbán
authors.affiliation: Tres Cantos Medicines Development Campus, GlaxoSmithKline, Severo Ochoa 2, ES-28760 Tres Cantos, Spain.
authors.forename: María Belén
authors.initials: MB
authors.lastname: Jiménez-Díaz
authors.affiliation: Tres Cantos Medicines Development Campus, GlaxoSmithKline, Severo Ochoa 2, ES-28760 Tres Cantos, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Viera-Morilla
authors.affiliation: Tres Cantos Medicines Development Campus, GlaxoSmithKline, Severo Ochoa 2, ES-28760 Tres Cantos, Spain.
authors.forename: Iñigo
authors.initials: I
authors.lastname: Angulo-Barturen
authors.affiliation: Nanoscience and Nanotechnology Institute (IN2UB), University of Barcelona, Martí i Franquès 1, ES-08028 Barcelona, Spain; Departament de Fisicoquímica, Facultat de Farmàcia, University of Barcelona, Av. Joan XXIII, s/n, ES-08028 Barcelona, Spain.
authors.forename: Maria Antònia
authors.initials: MA
authors.lastname: Busquets
authors.affiliation: Nanomalaria Group, Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 10-12, ES-08028 Barcelona, Spain; Barcelona Institute for Global Health (ISGlobal, Hospital Clínic-Universitat de Barcelona), Rosselló 149-153, ES-08036 Barcelona, Spain; Nanoscience and Nanotechnology Institute (IN2UB), University of Barcelona, Martí i Franquès 1, ES-08028 Barcelona, Spain. Electronic address: xfernandez_busquets@ub.edu.
authors.forename: Xavier
authors.initials: X
authors.lastname: Fernàndez-Busquets
mesh_terms: D000818:Animals; D000906:Antibodies; D000953:Antigens, Protozoan / Q000276:immunology; D000962:Antimalarials / Q000008:administration & dosage*; D002352:Carrier Proteins / Q000276:immunology; D002738:Chloroquine / Q000008:administration & dosage*; D016503:Drug Delivery Systems; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism / Q000469:parasitology; D005260:Female; D006801:Humans; D008081:Liposomes; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000517:prevention & control; D008565:Membrane Proteins; D008822:Mice, Transgenic; D009842:Oligopeptides / Q000276:immunology; D010963:Plasmodium falciparum / Q000187:drug effects / Q000254:growth & development; D011319:Primaquine / Q000008:administration & dosage*; D015800:Protozoan Proteins / Q000276:immunology
chemical_list: D000906:Antibodies; D000953:Antigens, Protozoan; D000962:Antimalarials; D002352:Carrier Proteins; C514457:HRP-2 antigen, Plasmodium falciparum; D008081:Liposomes; C478484:MAHRP-1 protein, Plasmodium falciparum; D008565:Membrane Proteins; D009842:Oligopeptides; D015800:Protozoan Proteins; D002738:Chloroquine; D011319:Primaquine
keywords: Chloroquine (PubChem CID: 2719); Cholesterol (PubChem CID: 5997); DOPC (PubChem CID: 6437081); DSPC (PubChem CID: 94190); Hoechst 33342 (PubChem CID: 1464); Immunoliposomes; Malaria; Maleimide (PubChem CID: 10935); Nanomedicine; Plasmodium; Primaquine (PubChem CID: 4908); Pyranine (PubChem CID: 61389); SATA (PubChem CID: 127532); Targeted drug delivery