Development of polyvalent erythrocyte- and parasitized erythrocyte-targeted nanovectors as novel site-specific drug delivery approaches for Plasmodium falciparum malaria chemotherapy

Abstract

Bearing in mind the absence of an effective preventive vaccine against malaria (WHO, 2015) and its severe clinical manifestations that are principally associated with red blood cell (RBC) destruction and parasitized-RBC (pRBC) cytoadherence to host cells causing in turn nearly half a million deaths every year, this disease represents nowadays a major threat to life and consequently its control and ultimate global eradication must be undertaken without preconceptions.

Additionally, the basic rationale followed by most of the currently marketed antimalarial approaches is based on the administration of single/multiple drugs on their own, strategy that promotes the emergence of drug-resistant parasites owing to the limitation in delivering drug payloads into the pRBC high enough to kill the intracellular parasite while at the same time minimizing the risk of causing toxic side effects to the patient. Such dichotomy has been successfully addressed through the preparation of RBC- and pRBC-targeted drug delivery systems in the form of antibody-vectorized liposomes (iLPs), among other types of nanovectors, improving in this way the activity of antimalarials (Chandra, 1991; Owais, 1995; Urbán, 2011).

Nevertheless, the aforesaid iLP models have been poorly characterized with a minimal knowledge regarding: their mechanism of interaction with the target cell, release kinetics of encapsulated material or antibody coupling yield; and their recognized antigens have not been reported or are still unknown. Besides, the improvement in drug activity they have provided has been rather modest when evaluated against the human-infecting Plasmodium falciparum species. The main scope of this PhD thesis has been in this regard the characterization and development of more effective immunoliposomal nanovectors against P. falciparum with special attention given to the obtainment of chemotherapeutic approaches displaying multiple mechanisms of action.

Antibody coupling yields of >40% were obtained by means of their derivatization with the SATA crosslinker and the incorporation of maleimide-containing, PEGylated phospholipids into LPs, which results in the highly stable thioether linkage. SATA/antibody molar ratios of up to 10× provided adequate antigen recognition and minimal iLP aggregation. Furthermore, the pH- driven active encapsulation of chloroquine (CQ) and primaquine antimalarials into DSPC-based LPs together with their conjugation with a monoclonal antibody specific for the glycophorin A antigen of RBCs (GPA-iLPs) enabled these iLPs (i) to completely recognize and become retained into both RBCs/pRBCs, as well as (ii) a total and stable drug encapsulation along with its effective intracellular release under parasite culture conditions. The improved antimalarial efficacy of CQ-loaded, GPA-iLPs was demonstrated in vivo in P. falciparum-infected, humanized mice through the reduction of their parasite densities to undetectable levels (<0.01% parasitemia) and following a 4 × 0.5 mg CQ/kg dosage schedule. Free CQ at a dosage 3.5 times higher was at least 40-fold less effective.

Moreover, lumefantrine-laden iLPs targeted against rosette-forming variants of PfEMP1 exhibited a great potential for severe malaria therapeutics by means of mechanically disrupting already generated rosettes while at the same time eliminating those parasites forming them. An increased activity of lumefantrine in reducing the number of rosette-forming pRBCs was obtained when delivered by homologous PfEMP1-targeted-iLPs with a ~5.5-fold decreased IC50 compared to either free drug or non-targeted LPs. The rosette-disrupting activity of anti- PfEMP1 antibodies was importantly preserved after their conjugation to LPs. Finally, based on a recent study in Toxoplasma gondii (Nagamune, 2008), the biosynthesis of ABA by P. falciparum was explored as a new target route for the design of antimalarials. However, ABA could not be found in late form-pRBC extracts even though the sensitivity for its detection had been extraordinarily improved (LOD of 0.03 ng/ml).