Exploration of sulfated polysaccharides as antimalarials and as targeting molecules for nanovector-mediated drug delivery to Plasmodium-infected cells

Abstract

Malaria is arguably one of the main medical concerns worldwide because of the numbers of people affected, the severity of the disease and the complexity of the life cycle of its causative agent, the protist Plasmodium spp. The clinical, social and economic burden of malaria has led to several waves of serious efforts to reach its control and eventual eradication, without success to this day. With the advent of nanoscience, renewed hopes have appeared of finally obtaining the long sought-after magic bullet against malaria in the form of a nanovector for the targeted delivery of antimalarial drugs exclusively to Plasmodium-infected cells. The research lines of this thesis were: the exploration of different types of encapsulating structure (liposomes and chitosan nanoparticles), targeting molecule (sulfated polysaccharides), and antimalarial compound (e.g. new structures derived from marine organisms) for the assembly of nanovectors capable of delivering their drug cargo with complete specificity to diseased cells.

Heparin had been demonstrated to have antimalarial activity and specific binding affinity for Plasmodium-infected red blood cells (pRBCs) vs. non-infected erythrocytes. For this reason, we have explored if both properties could be joined into a drug delivery strategy where heparin would have a dual role as antimalarial and as a targeting element of drug-loaded nanoparticles. Confocal fluorescence and transmission electron microscopy data show that after 30 min of being added to living pRBCs fluorescein-labeled heparin colocalizes with the intracellular parasites. Heparin electrostatically adsorbed onto positively charged liposomes containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane and loaded with the antimalarial drug primaquine was capable of increasing three-fold the activity of encapsulated drug in Plasmodium falciparum cultures. At concentrations below those inducing anticoagulation of mouse blood in vivo, parasiticidal activity was found to be the additive result of the separate activities of free heparin as antimalarial and of liposome-bound heparin as targeting element for encapsulated primaquine.

The significant antimalarial activity of heparin, against which there are no resistances known, has not been therapeutically exploited due to its potent anticoagulating activity. Marine organisms are a rich source of heparin-like sulfated polysaccharides whose potential medical use has been largely neglected. We have explored the antimalarial and anticoagulating activities of fucosylated chondroitin sulfates and fucans from the sea cucumbers Ludwigothurea grisea and Isostichopus badionotus, of a galactan from the red alga Botryocladia occidentalis, and of a glycan from the marine sponge Desmapsamma anchorata. In vitro assays reveal significant inhibition of P. falciparum growth for most polysaccharides that correlates with anticoagulating activity. The mechanism of this antimalarial activity operates through inhibition of erythrocyte invasion by Plasmodium, likely mediated by a coating of the parasite similar to that observed for heparin.

Although successful efforts have been made to obtain new nanostructures having affordable synthesis costs while exhibiting good performance in lowering the IC50 of drugs, new approaches are required to further optimize a scarcity of resources. In this regard, the adaptation of existing nanovector designs to new Plasmodium stages, antimalarial drugs, targeting molecules, or encapsulating structures is a strategy that can provide new cost-efficient therapies. Hence, we have explored the adaptation of different liposome prototypes that had been developed in our group for the delivery to pRBCs of the antimalarial drug primaquine. The results obtained indicate that the tuning of existing nanovessels to new malaria-related targets is a valid low-cost alternative to the development from scratch of new targeted nanosystems.

La malaria es una enfermedad aguda y/o crónica causada por protozoos del genero Plasmodium. Los parásitos son transmitidos de humano a humano a través de la picadura de mosquitos hembra de algunas especies de Anopheles previamente infectadas. Hay cinco especies que infectan a humanos: P. falciparum, P. vivax, P. ovale, P. malariae y P. knowlesi. Actualmente, P. vivax y P. falciparum son los más comúnmente encontrados.

Debido a la falta de especificidad con respecto a las células diana, las principales limitaciones de las estrategias quimioterapéuticas actuales son la administración de compuestos con poca o ninguna especificidad para los eritrocitos infectados, y la necesidad de administrar dosis totales altas que pueden representar efectos adversos para los pacientes. Por otro lado, la administración de dosis bajas conduce a la aparición de cepas de parásitos resistentes. Por estas razones, nos hemos centrado en la utilización de herramientas nanotecnológicas para tratar la malaria. Las principales ventajas de la nanotecnología en este campo son la entrega especifica de antimaláricos en las células diana lo que puede representar un aumento de la eficacia de los fármacos actuales y el posible uso de fármacos nuevos o ya conocidos de amplio espectro o medicamentos altamente tóxicos, disminuyendo así la aparición de resistencia.

Las principales razones por las que el parasito consigue escapar al sistema inmunitario del huésped son el hecho que los eritrocitos no son capaces de producir ni presentar antígenos; la alta variabilidad antigénica y la adhesión a células endoteliales de distintos órganos y tejidos. La adhesión a las células endoteliales representa un papel importante en el desenlace fatal de la malaria severa y se cree que es un proceso mediado por la proteína PfEMP1. Curiosamente, algunos polisacáridos sulfatados han demostrado actuar como moléculas de unión a pRBCs que puede o bien inhibir el secuestro de pRBCs, impedir la formación de rosetas o bloquear la adhesión de esporozoítos a los hepatocitos. Estas evidencias nos inspiraron a estudiar diversos polisacáridos sulfatados como moléculas diana y como antimaláricos. Además, nos hemos enfocado en la funcionalización de liposomas (conteniendo droga) con heparina como prueba de concepto y evaluado su actividad y mecanismo de acción en los estadios asexuales del parásito.