Ernest Moles

Nanotools for the Delivery of Antimicrobial Peptides

Antimicrobial peptide drugs are increasingly attractive therapeutic agents as their roles in physiopathological processes are being unraveled and because the development of recombinant DNA technology has made them economically affordable in large amounts and high purity. However, due to lack of specificity regarding the target cells, difficulty in attaining them, or reduced half-lives, most current administration methods require high doses. On the other hand, reduced specificity of toxic drugs demands low concentrations to minimize undesirable side-effects, thus incurring the risk of having sublethal amounts which favour the appearance of resistant microbial strains. In this scenario, targeted delivery can fulfill the objective of achieving the intake of total quantities sufficiently low to be innocuous for the patient but that locally are high enough to be lethal for the infectious agent. One of the major advances in recent years has been the size reduction of drug carriers that have dimensions in the nanometer scale and thus are much smaller than -and capable of being internalized by- many types of cells. Among the different types of potential antimicrobial peptide-encapsulating structures reviewed here are liposomes, dendritic polymers, solid core nanoparticles, carbon nanotubes, and DNA cages. These nanoparticulate systems can be functionalized with a plethora of biomolecules providing specificity of binding to particular cell types or locations; as examples of these targeting elements we will present antibodies, DNA aptamers, cell-penetrating peptides, and carbohydrates. Multifunctional Trojan horse-like nanovessels can be engineered by choosing the adequate peptide content, encapsulating structure, and targeting moiety for each particular application.

Immunoliposome-mediated drug delivery to Plasmodium-infected and non-infected red blood cells as a dual therapeutic/prophylactic antimalarial strategy

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.

Application of heparin as a dual agent with antimalarial and liposome targeting activities toward Plasmodium-infected red blood cells

UNLABELLED Heparin had been demonstrated to have antimalarial activity and specific binding affinity for Plasmodium-infected red blood cells (pRBCs) vs. non-infected erythrocytes. Here 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. FROM THE CLINICAL EDITOR Malaria remains an enormous global public health concern. In this study, a novel functionalized heparin formulation used as drug delivery agent for primaquine was demonstrated to result in threefold increased drug activity in cell cultures, and in a murine model it was able to provide these benefits in concentrations below what would be required for anticoagulation. Further studies are needed determine if this approach is applicable in the human disease as well.

Rosette-Disrupting Effect of an Anti-Plasmodial Compound for the Potential Treatment of Plasmodium falciparum Malaria Complications

The spread of artemisinin-resistant parasites could lead to higher incidence of patients with malaria complications. However, there are no current treatments that directly dislodge sequestered parasites from the microvasculature. We show that four common antiplasmodial drugs do not disperse rosettes (erythrocyte clusters formed by malaria parasites) and therefore develop a cell-based high-throughput assay to identify potential rosette-disrupting compounds. A pilot screen of 2693 compounds identified Malaria Box compound MMV006764 as a potential candidate. Although it reduced rosetting by a modest 20%, MMV006764 was validated to be similarly effective against both blood group O and A rosettes of three laboratory parasite lines. Coupled with its antiplasmodial activity and drug-likeness, MMV006764 represents the first small-molecule compound that disrupts rosetting and could potentially be used in a resource-limited setting to treat patients deteriorating rapidly from malaria complications. Such dual-action drugs that simultaneously restore microcirculation and reduce parasite load could significantly reduce malaria morbidity and mortality.

Development of drug-loaded immunoliposomes for the selective targeting and elimination of rosetting Plasmodium falciparum-infected red blood cells.

Parasite proteins exported to the surface of Plasmodium falciparum-parasitized red blood cells (pRBCs) have a major role in severe malaria clinical manifestation, where pRBC cytoadhesion and rosetting processes have been strongly linked with microvascular sequestration while avoiding both spleen filtration and immune surveillance. The parasite-derived and pRBC surface-exposed PfEMP1 protein has been identified as one of the responsible elements for rosetting and, therefore, considered as a promising vaccine candidate for the generation of rosette-disrupting antibodies against severe malaria. However, the potential role of anti-rosetting antibodies as targeting molecules for the functionalization of antimalarial drug-loaded nanovectors has never been studied. Our manuscript presents a proof-of-concept study where the activity of an immunoliposomal vehicle with a dual performance capable of specifically recognizing and disrupting rosettes while simultaneously eliminating those pRBCs forming them has been assayed in vitro. A polyclonal antibody against the NTS-DBL1α N-terminal domain of a rosetting PfEMP1 variant has been selected as targeting molecule and lumefantrine as the antimalarial payload. After 30min incubation with 2μM encapsulated drug, a 70% growth inhibition for all parasitic forms in culture (IC50: 414nM) and a reduction in ca. 60% of those pRBCs with a rosetting phenotype (IC50: 747nM) were achieved. This immunoliposomal approach represents an innovative combination therapy for the improvement of severe malaria therapeutics having a broader spectrum of activity than either anti-rosetting antibodies or free drugs on their own.

Loading antimalarial drugs into noninfected red blood cells: an undesirable roommate for Plasmodium

Ernest Moles Nanomalaria Unit, Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 10–12, Barcelona ES08028, Spain and Barcelona Institute for Global Health (ISGlobal, Barcelona Ctr. Int. Health Res. [CRESIB], Hospital Clinic-Universitat de Barcelona), Rossello 149–153, Barcelona ES-08036, Spain and Nanoscience & Nanotechnology Institute (INUB), University of Barcelona, Marti i Franques 1, Barcelona ES-08028, Spain

Possible roles of amyloids in malaria pathophysiology

The main therapeutic and prophylactic tools against malaria have been locked for more than a century in the classical approaches of using drugs targeting metabolic processes of the causing agent, the protist Plasmodium spp., and of designing vaccines against chosen antigens found on the parasites surface. Given the extraordinary resources exhibited by Plasmodium to escape these traditional strategies, which have not been able to free humankind from the scourge of malaria despite much effort invested in them, new concepts have to be explored in order to advance toward eradication of the disease. In this context, amyloid-forming proteins and peptides found in the proteome of the pathogen should perhaps cease being regarded as mere anomalous molecules. Their likely functionality in the pathophysiology of Plasmodium calls for attention being paid to them as a possible Achilles’ heel of malaria. Here we will give an overview of Plasmodium-encoded amyloid-forming polypeptides as potential therapeutic targets and toxic elements, particularly in relation to cerebral malaria and the blood–brain barrier function. We will also discuss the recent finding that the genome of the parasite contains an astonishingly high proportion of prionogenic domains.

2-picolylamine derivatization for high sensitivity detection of abscisic acid in apicomplexan blood-infecting parasites

We have developed a new liquid chromatography-electrospray ionization tandem mass spectrometry methodology based on 2-picolylamine derivatization and positive ion mode detection for abscisic acid (ABA) identification. The selected reaction leads to the formation of an amide derivative which contains a highly active pyridyl group. The enhanced ionization allows for a 700-fold increase over commonly monitored unmodified ABA, which in turn leads to excellent limits of detection and quantification values of 0.03 and 0.15ngmL-1, respectively. This method has been validated in the highly complex matrix of a red blood cell extract. In spite of the high sensitivity achieved, ABA could not be detected in Plasmodium falciparum-infected red blood cells, suggesting that, if present, it will be found either in ultratrace amounts or as brief bursts at defined time points within the intraerythrocytic cycle and/or in the form of a biosynthetic analogue.