by Keyword: Plasmodium

By year:[ 2021 | 2020 | 2019 | 2018 | 2017 | 2016 | 2015 | 2014 | 2013 | 2012 | 2011 | 2010 | 2009 | 2008 | 2007 | 2006 | 2005 ]

Fonte, M., Tassi, N., Fontinha, D., Bouzón-Arnáiz, I., Ferraz, R., Araújo, M. J., Fernàndez-Busquets, X., Prudêncio, M., Gomes, P., Teixeira, C., (2021). 4,9-Diaminoacridines and 4-aminoacridines as dual-stage antiplasmodial hits ChemMedChem 16, (5), 788-792

Multi-stage drugs have been prioritized in antimalarial drug discovery, as targeting more than one process in the Plasmodium life cycle is likely to increase efficiency, while decreasing the chances of emergence of resistance by the parasite. Herein, we disclose two novel acridine-based families of compounds that combine the structural features of primaquine and chloroquine. Compounds prepared and studied thus far retained the in vitro activity displayed by the parent drugs against the erythrocytic stages of chloroquine-sensitive and -resistant Plasmodium falciparum strains, and against the hepatic stages of Plasmodium berghei, hence acting as dual-stage antiplasmodial hits.

Keywords: Acridines, Antimalarial activity, Blood-stage, Liver-stage, Malaria, Plasmodium, Synthesis

de la Serna, E., Arias-Alpízar, K., Borgheti-Cardoso, L. N., Sanchez-Cano, A., Sulleiro, E., Zarzuela, F., Bosch-Nicolau, P., Salvador, F., Molina, I., Ramírez, M., Fernàndez-Busquets, X., Sánchez-Montalvá, A., Baldrich, E., (2021). Detection of Plasmodium falciparum malaria in 1 h using a simplified enzyme-linked immunosorbent assay Analytica Chimica Acta 1152

Malaria is a parasitic disease caused by protists of the genus Plasmodium, which are transmitted to humans through the bite of infected female Anopheles mosquitoes. Analytical methodologies and efficient drugs exist for the early detection and treatment of malaria, and yet this disease continues infecting millions of people and claiming several hundred thousand lives each year. One of the reasons behind this failure to control the disease is that the standard method for malaria diagnosis, microscopy, is time-consuming and requires trained personnel. Alternatively, rapid diagnostic tests, which have become common for point-of-care testing thanks to their simplicity of use, tend to be insufficiently sensitive and reliable, and PCR, which is sensitive, is too complex and expensive for massive population screening. In this work, we report a sensitive simplified ELISA for the quantitation of Plasmodium falciparum lactate dehydrogenase (Pf-LDH), which is capable of detecting malaria in 45–60 min. Assay development was founded in the selection of high-performance antibodies, implementation of a poly-horseradish peroxidase (polyHRP) signal amplifier, and optimization of whole-blood sample pre-treatment. The simplified ELISA achieved limits of detection (LOD) and quantification (LOQ) of 0.11 ng mL−1 and 0.37 ng mL−1, respectively, in lysed whole blood, and an LOD comparable to that of PCR in Plasmodium in vitro cultures (0.67 and 1.33 parasites μL−1 for ELISA and PCR, respectively). Accordingly, the developed immunoassay represents a simple and effective diagnostic tool for P. falciparum malaria, with a time-to-result of <60 min and sensitivity similar to the reference PCR, but easier to implement in low-resource settings. © 2021 Elsevier B.V.

Keywords: Malaria quantitative diagnosis, Plasmodium culture, Plasmodium LDH, polyHRP signal amplifier, Simplified ELISA

Biosca, A., Cabanach, P., Abdulkarim, M., Gumbleton, M., Gómez-Canela, C., Ramírez, M., Bouzón-Arnáiz, I., Avalos-Padilla, Y., Borros, S., Fernàndez-Busquets, X., (2021). Zwitterionic self-assembled nanoparticles as carriers for Plasmodium targeting in malaria oral treatment Journal of Controlled Release 331, 364-375

The current decline in antimalarial drug efficacy due to the evolution of resistant Plasmodium strains calls for new strategies capable of improving the bioavailability of antimalarials, especially of those whose lipophilic character imparts them a low solubility in biological fluids. Here we have designed, synthesized and characterized amphiphilic zwitterionic block copolymers forming nanoparticles capable of penetrating the intestinal epithelium that can be used for oral administration. Poly(butyl methacrylate-co-morpholinoethyl sulfobetaine methacrylate) (PBMA-MESBMA)-based nanoparticles exhibited a specific targeting to Plasmodium falciparum-infected vs. parasite-free red blood cells (74.8%/0.8% respectively), which was maintained upon encapsulation of the lipophilic antimalarial drug curcumin (82.6%/0.3%). The in vitro efficacy of curcumin upon encapsulation was maintained relative to the free compound, with an IC50 around 5 μM. In vivo assays indicated a significantly increased curcumin concentration in the blood of mice one hour after being orally fed PBMA-MESBMA-curcumin in comparison to the administration of free drug (18.7 vs. 2.1 ng/ml, respectively). At longer times, however, plasma curcumin concentration equaled between free and encapsulated drug, which was reflected in similar in vivo antimalarial activities in Plasmodium yoelii yoelii-infected mice. Microscopic analysis in blood samples of fluorescently labeled PBMA-MESBMA revealed the presence of the polymer inside P. yoelii yoelii-parasitized erythrocytes one hour after oral administration to infected animals. © 2021 Elsevier B.V.

Keywords: Curcumin, Drug delivery, Malaria, PBMA-MESBMA, Plasmodium, Zwitterionic block copolymers

Ruiz-Vega, G., Arias-Alpízar, K., de la Serna, E., Borgheti-Cardoso, L. N., Sulleiro, E., Molina, I., Fernàndez-Busquets, X., Sánchez-Montalvá, A., del Campo, F. J., Baldrich, E., (2020). Electrochemical POC device for fast malaria quantitative diagnosis in whole blood by using magnetic beads, Poly-HRP and microfluidic paper electrodes Biosensors and Bioelectronics 150, 111925

Malaria, a parasitic infection caused by Plasmodium parasites and transmitted through the bite of infected female Anopheles mosquitos, is one of the main causes of mortality in many developing countries. Over 200 million new infections and nearly half a million deaths are reported each year, and more than three billion people are at risk of acquiring malaria worldwide. Nevertheless, most malaria cases could be cured if detected early. Malaria eradication is a top priority of the World Health Organisation. However, achieving this goal will require mass population screening and treatment, which will be hard to accomplish with current diagnostic tools. We report an electrochemical point-of-care device for the fast, simple and quantitative detection of Plasmodium falciparum lactate dehydrogenase (PfLDH) in whole blood samples. Sample analysis includes 5-min lysis to release intracellular parasites, and stirring for 5 more min with immuno-modified magnetic beads (MB) along with an immuno-modified signal amplifier. The rest of the magneto-immunoassay, including sample filtration, MB washing and electrochemical detection, is performed at a disposable paper electrode microfluidic device. The sensor provides PfLDH quantitation down to 2.47 ng mL−1 in spiked samples and for 0.006–1.5% parasitemias in Plasmodium-infected cultured red blood cells, and discrimination between healthy individuals and malaria patients presenting parasitemias >0.3%. Quantitative malaria diagnosis is attained with little user intervention, which is not achieved by other diagnostic methods.

Keywords: Electrochemical magneto-immunosensor, Malaria quantitative diagnosis, Paper microfluidic electrode, Plasmodium LDH, Point-of-care (POC) testing

Borgheti-Cardoso, L. N., Kooijmans, S. A. A., Gutiérrez Chamorro, L., Biosca, A., Lantero, E., Ramírez, M., Avalos-Padilla, Y., Crespo, I., Fernández, I., Fernandez-Becerra, C., del Portillo, H. A., Fernàndez-Busquets, X., (2020). Extracellular vesicles derived from Plasmodium-infected and non-infected red blood cells as targeted drug delivery vehicles International Journal of Pharmaceutics 587, 119627

Among several factors behind drug resistance evolution in malaria is the challenge of administering overall doses that are not toxic for the patient but that, locally, are sufficiently high to rapidly kill the parasites. Thus, a crucial antimalarial strategy is the development of drug delivery systems capable of targeting antimalarial compounds to Plasmodium with high specificity. In the present study, extracellular vesicles (EVs) have been evaluated as a drug delivery system for the treatment of malaria. EVs derived from naive red blood cells (RBCs) and from Plasmodium falciparum-infected RBCs (pRBCs) were isolated by ultrafiltration followed by size exclusion chromatography. Lipidomic characterization showed that there were no significant qualitative differences between the lipidomic profiles of pRBC-derived EVs (pRBC-EVs) and RBC-derived EVs (RBC-EVs). Both EVs were taken up by RBCs and pRBCs, although pRBC-EVs were more efficiently internalized than RBC-EVs, which suggested their potential use as drug delivery vehicles for these cells. When loaded into pRBC-EVs, the antimalarial drugs atovaquone and tafenoquine inhibited in vitro P. falciparum growth more efficiently than their free drug counterparts, indicating that pRBC-EVs can potentially increase the efficacy of several small hydrophobic drugs used for the treatment of malaria.

Keywords: Antimalarial drugs, Drug delivery, Extracellular vesicles, Malaria, Plasmodium falciparum

Lantero, E., Fernandes, J., Aláez-Versón, C. R., Gomes, J., Silveira, H., Nogueira, F., Fernàndez-Busquets, X., (2020). Heparin administered to anopheles in membrane feeding assays blocks plasmodium development in the mosquito Biomolecules 10, (8), 1136

Innovative antimalarial strategies are urgently needed given the alarming evolution of resistance to every single drug developed against Plasmodium parasites. The sulfated glycosaminoglycan heparin has been delivered in membrane feeding assays together with Plasmodium berghei-infected blood to Anopheles stephensi mosquitoes. The transition between ookinete and oocyst pathogen stages in the mosquito has been studied in vivo through oocyst counting in dissected insect midguts, whereas ookinete interactions with heparin have been followed ex vivo by flow cytometry. Heparin interferes with the parasite’s ookinete–oocyst transition by binding ookinetes, but it does not affect fertilization. Hypersulfated heparin is a more efficient blocker of ookinete development than native heparin, significantly reducing the number of oocysts per midgut when offered to mosquitoes at 5 µg/mL in membrane feeding assays. Direct delivery of heparin to mosquitoes might represent a new antimalarial strategy of rapid implementation, since it would not require clinical trials for its immediate deployment.

Keywords: Anopheles, Antimalarial drugs, Heparin, Malaria, Mosquito, Ookinete, Plasmodium, Transmission blocking

Biosca, A., Dirscherl, L., Moles, E., Imperial, S., Fernàndez-Busquets, X., (2019). An immunoPEGliposome for targeted antimalarial combination therapy at the nanoscale Pharmaceutics 11, (7), 341

Combination therapies, where two drugs acting through different mechanisms are administered simultaneously, are one of the most efficient approaches currently used to treat malaria infections. However, the different pharmacokinetic profiles often exhibited by the combined drugs tend to decrease treatment efficacy as the compounds are usually eliminated from the circulation at different rates. To circumvent this obstacle, we have engineered an immunoliposomal nanovector encapsulating hydrophilic and lipophilic compounds in its lumen and lipid bilayer, respectively. The antimalarial domiphen bromide has been encapsulated in the liposome membrane with good efficiency, although its high IC50 of ca. 1 μM for living parasites complicates its use as immunoliposomal therapy due to erythrocyte agglutination. The conjugation of antibodies against glycophorin A targeted the nanocarriers to Plasmodium-infected red blood cells and to gametocytes, the sole malaria parasite stage responsible for the transmission from the human to the mosquito vector. The antimalarials pyronaridine and atovaquone, which block the development of gametocytes, have been co-encapsulated in glycophorin A-targeted immunoliposomes. The co-immunoliposomized drugs have activities significantly higher than their free forms when tested in in vitro Plasmodium falciparum cultures: Pyronaridine and atovaquone concentrations that, when encapsulated in immunoliposomes, resulted in a 50% inhibition of parasite growth had no effect on the viability of the pathogen when used as free drugs.

Keywords: Combination therapy, Immunoliposomes, Malaria, Nanomedicine, Nanotechnology, Plasmodium, Targeted drug delivery

Aguiar, L., Biosca, A., Lantero, E., Gut, J., Vale, N., Rosenthal, P. J., Nogueira, F., Andreu, D., Fernàndez-Busquets, X., Gomes, P., (2019). Coupling the antimalarial cell penetrating peptide TP10 to classical antimalarial drugs primaquine and chloroquine produces strongly hemolytic conjugates Molecules 24, (24), 4559

Recently, we disclosed primaquine cell penetrating peptide conjugates that were more potent than parent primaquine against liver stage Plasmodium parasites and non-toxic to hepatocytes. The same strategy was now applied to the blood-stage antimalarial chloroquine, using a wide set of peptides, including TP10, a cell penetrating peptide with intrinsic antiplasmodial activity. Chloroquine-TP10 conjugates displaying higher antiplasmodial activity than the parent TP10 peptide were identified, at the cost of an increased hemolytic activity, which was further confirmed for their primaquine analogues. Fluorescence microscopy and flow cytometry suggest that these drug-peptide conjugates strongly bind, and likely destroy, erythrocyte membranes. Taken together, the results herein reported put forward that coupling antimalarial aminoquinolines to cell penetrating peptides delivers hemolytic conjugates. Hence, despite their widely reported advantages as carriers for many different types of cargo, from small drugs to biomacromolecules, cell penetrating peptides seem unsuitable for safe intracellular delivery of antimalarial aminoquinolines due to hemolysis issues. This highlights the relevance of paying attention to hemolytic effects of cell penetrating peptide-drug conjugates.

Keywords: Antimalarial, Cell penetrating peptide, Chloroquine, Erythrocyte fluorescence, Flow cytometry, Hemolysis, Microscopy, Plasmodium, Primaquine, Red blood cell

Martí Coma-Cros, E., Biosca, A., Marques, J., Carol, L., Urbán, P., Berenguer, D., Riera, M. C., Delves, M., Sinden, R. E., Valle-Delgado, J. J., Spanos, L., Siden-Kiamos, I., Pérez, P., Paaijmans, K., Rottmann, M., Manfredi, A., Ferruti, P., Ranucci, E., Fernàndez-Busquets, X., (2018). Polyamidoamine nanoparticles for the oral administration of antimalarial drugs Pharmaceutics 10, (4), 225

Current strategies for the mass administration of antimalarial drugs demand oral formulations to target the asexual Plasmodium stages in the peripheral bloodstream, whereas recommendations for future interventions stress the importance of also targeting the transmission stages of the parasite as it passes between humans and mosquitoes. Orally administered polyamidoamine (PAA) nanoparticles conjugated to chloroquine reached the blood circulation and cured Plasmodium yoelii-infected mice, slightly improving the activity of the free drug and inducing in the animals immunity against malaria. Liquid chromatography with tandem mass spectrometry analysis of affinity chromatography-purified PAA ligands suggested a high adhesiveness of PAAs to Plasmodium falciparum proteins, which might be the mechanism responsible for the preferential binding of PAAs to Plasmodium-infected erythrocytes vs. non-infected red blood cells. The weak antimalarial activity of some PAAs was found to operate through inhibition of parasite invasion, whereas the observed polymer intake by macrophages indicated a potential of PAAs for the treatment of certain coinfections such as Plasmodium and Leishmania. When fluorescein-labeled PAAs were fed to females of the malaria mosquito vectors Anopheles atroparvus and Anopheles gambiae, persistent fluorescence was observed in the midgut and in other insect’s tissues. These results present PAAs as a versatile platform for the encapsulation of orally administered antimalarial drugs and for direct administration of antimalarials to mosquitoes, targeting mosquito stages of Plasmodium.

Keywords: Anopheles, Antimalarial drugs, Malaria, Mosquitoes, Nanomedicine, Nanotechnology, Plasmodium, Polyamidoamines, Polymers, Targeted drug delivery

Pallarès, Irantzu, de Groot, Natalia S., Iglesias, Valentín, Sant'Anna, Ricardo, Biosca, Arnau, Fernàndez-Busquets, Xavier, Ventura, Salvador, (2018). Discovering putative prion-like proteins in Plasmodium falciparum: A computational and experimental analysis Frontiers in Microbiology 9, Article 1737

Prions are a singular subset of proteins able to switch between a soluble conformation and a self-perpetuating amyloid state. Traditionally associated with neurodegenerative diseases, increasing evidence indicates that organisms exploit prion-like mechanisms for beneficial purposes. The ability to transit between conformations is encoded in the so-called prion domains, long disordered regions usually enriched in glutamine/asparagines residues. Interestingly, Plasmodium falciparum, the parasite that causes the most virulent form of malaria, is exceptionally rich in proteins bearing long Q/N-rich sequence stretches, accounting for roughly 30% of the proteome. This biased composition suggests that these protein regions might correspond to prion-like domains (PrLDs) and potentially form amyloid assemblies. To investigate this possibility, we performed a stringent computational survey for Q/N-rich PrLDs on P. falciparum. Our data indicate that ~10% of P. falciparum protein sequences have prionic signatures, and that this subproteome is enriched in regulatory proteins, such as transcription factors and RNA-binding proteins. Furthermore, we experimentally demonstrate for several of the identified PrLDs that, despite their disordered nature, they contain inner short sequences able to spontaneously self-assemble into amyloid-like structures. Although the ability of these sequences to nucleate the conformational conversion of the respective full-length proteins should still be demonstrated, our analysis suggests that, as previously described for other organisms, prion-like proteins might also play a functional role in P. falciparum.

Keywords: Plasmodium, Protein aggregation, Amyloid, Prion, Q-N-rich sequences, Protein Disorder

Martí Coma-Cros, Elisabet, Biosca, Arnau, Lantero, Elena, Manca, Maria, Caddeo, Carla, Gutiérrez, Lucía, Ramírez, Miriam, Borgheti-Cardoso, Livia, Manconi, Maria, Fernàndez-Busquets, Xavier, (2018). Antimalarial activity of orally administered curcumin incorporated in Eudragit®-containing liposomes International Journal of Molecular Sciences 19, (5), 1361

Curcumin is an antimalarial compound easy to obtain and inexpensive, having shown little toxicity across a diverse population. However, the clinical use of this interesting polyphenol has been hampered by its poor oral absorption, extremely low aqueous solubility and rapid metabolism. In this study, we have used the anionic copolymer Eudragit® S100 to assemble liposomes incorporating curcumin and containing either hyaluronan (Eudragit-hyaluronan liposomes) or the water-soluble dextrin Nutriose® FM06 (Eudragit-nutriosomes). Upon oral administration of the rehydrated freeze-dried nanosystems administered at 25/75 mg curcumin·kg−1·day−1, only Eudragit-nutriosomes improved the in vivo antimalarial activity of curcumin in a dose-dependent manner, by enhancing the survival of all Plasmodium yoelii-infected mice up to 11/11 days, as compared to 6/7 days upon administration of an equal dose of the free compound. On the other hand, animals treated with curcumin incorporated in Eudragit-hyaluronan liposomes did not live longer than the controls, a result consistent with the lower stability of this formulation after reconstitution. Polymer-lipid nanovesicles hold promise for their development into systems for the oral delivery of curcumin-based antimalarial therapies.

Keywords: Malaria, Curcumin, Nanomedicine, Oral administration, Lipid nanovesicles, Eudragit, Nutriose, Hyaluronan, Plasmodium yoelii

Borgheti-Cardoso, L.N., Fernàndez-Busquets, X., (2018). Turning Plasmodium survival strategies against itself Future Medicinal Chemistry 10, (19), 2245-2248

Moles, E., Galiano, S., Gomes, A., Quiliano, M., Teixeira, C., Aldana, I., Gomes, P., Fernàndez-Busquets, X., (2017). ImmunoPEGliposomes for the targeted delivery of novel lipophilic drugs to red blood cells in a falciparum malaria murine model Biomaterials 145, 178-191

Most drugs currently entering the clinical pipeline for severe malaria therapeutics are of lipophilic nature, with a relatively poor solubility in plasma and large biodistribution volumes. Low amounts of these compounds do consequently accumulate in circulating Plasmodium-infected red blood cells, exhibiting limited antiparasitic activity. These drawbacks can in principle be satisfactorily dealt with by stably encapsulating drugs in targeted nanocarriers. Here this approach has been adapted for its use in immunocompetent mice infected by the Plasmodium yoelii 17XL lethal strain, selected as a model for human blood infections by Plasmodium falciparum. Using immunoliposomes targeted against a surface protein characteristic of the murine erythroid lineage, the protocol has been applied to two novel antimalarial lipophilic drug candidates, an aminoquinoline and an aminoalcohol. Large encapsulation yields of >90% were obtained using a citrate-buffered pH gradient method and the resulting immunoliposomes reached in vivo erythrocyte targeting and retention efficacies of >80%. In P. yoelii-infected mice, the immunoliposomized aminoquinoline succeeded in decreasing blood parasitemia from severe to uncomplicated malaria parasite densities (i.e. from ≥25% to ca. 5%), whereas the same amount of drug encapsulated in non-targeted liposomes had no significant effect on parasite growth. Pharmacokinetic analysis indicated that this good performance was obtained with a rapid clearance of immunoliposomes from the circulation (blood half-life of ca. 2 h), suggesting a potential for improvement of the proposed model.

Keywords: Immunoliposomes, Malaria, Nanomedicine, Plasmodium falciparum, Plasmodium yoelii 17XL, Targeted drug delivery

Marques, J., Valle-Delgado, J. J., Urbán, P., Baró, E., Prohens, R., Mayor, A., Cisteró, P., Delves, M., Sinden, R. E., Grandfils, C., de Paz, J. L., García-Salcedo, J. A., Fernàndez-Busquets, X., (2017). Adaptation of targeted nanocarriers to changing requirements in antimalarial drug delivery Nanomedicine: Nanotechnology, Biology, and Medicine 13, (2), 515-525

The adaptation of existing antimalarial nanocarriers to new Plasmodium stages, drugs, targeting molecules, or encapsulating structures is a strategy that can provide new nanotechnology-based, cost-efficient therapies against malaria. We have explored the modification of different liposome prototypes that had been developed in our group for the targeted delivery of antimalarial drugs to Plasmodium-infected red blood cells (pRBCs). These new models include: (i) immunoliposome-mediated release of new lipid-based antimalarials; (ii) liposomes targeted to pRBCs with covalently linked heparin to reduce anticoagulation risks; (iii) adaptation of heparin to pRBC targeting of chitosan nanoparticles; (iv) use of heparin for the targeting of Plasmodium stages in the mosquito vector; and (v) use of the non-anticoagulant glycosaminoglycan chondroitin 4-sulfate as a heparin surrogate for pRBC targeting. The results presented indicate that the tuning of existing nanovessels to new malaria-related targets is a valid low-cost alternative to the de novo development of targeted nanosystems.

Keywords: Glycosaminoglycans, Malaria, Nanomedicine, Plasmodium, Targeted drug delivery

Aláez-Versón, C. R., Lantero, E., Fernàndez-Busquets, X., (2017). Heparin: New life for an old drug Nanomedicine 12, (14), 1727-1744

Heparin is one of the oldest drugs, which nevertheless remains in widespread clinical use as an inhibitor of blood coagulation. The history of its identification a century ago unfolded amid one of the most fascinating scientific controversies turning around the distribution of credit for its discovery. The composition, purification and structure-function relationship of this naturally occurring glycosaminoglycan regarding its classical role as anticoagulant will be dealt with before proceeding to discuss its therapeutic potential in, among other, inflammatory and infectious disease, cancer treatment, cystic fibrosis and Alzheimer's disease. The first bibliographic reference hit using the words 'nanomedicine' and 'heparin' is as recent as 2008. Since then, nanomedical applications of heparin have experienced an exponential growth that will be discussed in detail, with particular emphasis on its antimalarial activity. Some of the most intriguing potential applications of heparin nanomedicines will be exposed, such as those contemplating the delivery of drugs to the mosquito stages of malaria parasites.

Keywords: Anopheles, Antimalarial drugs, Heparin, Malaria, Mosquitoes, Nanomedicine, Nanotechnology, Plasmodium, Targeted drug delivery

Moles, E., Marcos, J., Imperial, S., Pozo, O. J., Fernàndez-Busquets, X., (2017). 2-picolylamine derivatization for high sensitivity detection of abscisic acid in apicomplexan blood-infecting parasites Talanta 168, 130-135

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 0.15 ng mL-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.

Keywords: Abscisic acid, Apicomplexa, Liquid chromatography-electrospray ionization tandem mass spectrometry, Malaria, Picolylamine, Plasmodium falciparum

Fernàndez-Busquets, X., (2016). Novel strategies for Plasmodium-targeted drug delivery Expert Opinion on Drug Delivery , 13, (7), 919-922

Valle-Delgado, J. J., Fernàndez-Busquets, X., (2016). Rapid diagnostic tests for malaria: Past, present and future Future Microbiology , 11, (11), 1379-1382

Moles, E., Urbán, P., Jiménez-Díaz, M. B., Viera-Morilla, S., Angulo-Barturen, I., Busquets, M. A., Fernàndez-Busquets, X., (2015). Immunoliposome-mediated drug delivery to Plasmodium-infected and non-infected red blood cells as a dual therapeutic/prophylactic antimalarial strategy Journal of Controlled Release 210, 217-229

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 0.5 μM total lipid in the culture, with >95% of added liposomes retained on cell surfaces. When exposed for only 15 min to Plasmodium falciparum in vitro cultures of early stages, free CQ had no significant effect on the viability of the parasite up to 200 nM, whereas immunoliposomal 50 nM CQ completely arrested its growth. In vivo assays in mice showed that immunoliposomes cleared the pathogen below detectable levels at a CQ dose of 0.5 mg/kg, whereas free CQ administered at 1.75 mg/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.

Keywords: Immunoliposomes, Malaria, Nanomedicine, Plasmodium, Targeted drug delivery

Moles, E., Fernàndez-Busquets, X., (2015). Loading antimalarial drugs into noninfected red blood cells: An undesirable roommate for Plasmodium Future Medicinal Chemistry 7, (7), 837-840

The malaria parasite, Plasmodium spp., is a delicate unicellular organism unable to survive in free form for more than a couple of minutes in the bloodstream. Upon injection in a human by its Anopheles mosquito vector, Plasmodium sporozoites pass through the liver with the aim of invading hepatocytes. Those which succeed spend inside their host cell a recovery time before replicating and entering the blood circulation as fragile merozoites, although their exposure to host defenses is extraordinarily short. Quick invasion of red blood cells (RBCs) in a process lasting just a few minutes allows the parasite to escape immune system surveillance. For most of its erythrocytic cycle the pathogen feeds mainly on hemoglobin as it progresses from the early blood stages, termed rings, to the late forms trophozoites and schizonts. Early stages are ideal targets for antimalarial therapies because drugs delivered to them would have a longer time to kill the parasite before it completes its development. However, only 6 h after invasion does the permeability of the infected erythrocyte to anions and small nonelectrolytes, including some drugs, start to increase as the parasite matures [1]. During this maturation process the parasite hydrolyzes hemoglobin in a digestive vacuole, which is the target of many amphiphilic drugs that freely cross the RBC membrane and accumulate intracellularly. As a result, most antimalarials start affecting the infected cell relatively late in the intraerythrocytic parasite life cycle, when their effect is probably often too short to be lethal to Plasmodium.

Keywords: Malaria, Nanomedicine, Plasmodium, Red blood cell, Targeted drug delivery

Pujol, A., Urbán, P., Riera, C., Fisa, R., Molina, I., Salvador, F., Estelrich, J., Fernàndez-Busquets, X., (2014). Application of quantum dots to the study of liposome targeting in leishmaniasis and malaria International Journal of Theoretical and Applied Nanotechnology , 2, (1), 1-8

Nanotechnological devices for therapeutic applications are massively addressed to diseases prevalent in the developed world, particularly cancer, because of the wrong assumption (for both ethical and technical reasons) that nanomedicines are too expensive and thus they can not be applied to diseases of poverty. Here we have applied quantum dots to study at the cellular level the delivery of the contents of liposomes to erythrocytes infected by the malaria parasite Plasmodium falciparum, and to macrophages infected by the leishmaniasis causative agent Leishmania infantum. A number of works have reported on the encapsulation in liposomes of drugs against both diseases as a strategy to increase therapeutic efficacy and decrease unspecific toxicity. Liposome-carried drugs end up inside Plasmodium-infected red blood cells (pRBCs) and in the phagolysosome system of Leishmania-infected macrophages but some knowledge gaps still obscure subcellular events related to these processes. As a proof of concept, we have used confocal fluorescence microscopy to follow the fate in pRBCs and infected macrophages of quantum dots encapsulated in liposomes, and of lysosomes, leishmaniasis and malaria parasites, nuclei, and phagosomes. Our data indicate that liposomes merge their lipid bilayers with pRBC plasma membranes but are engulfed by macrophages, where they fuse with lysosomes. Lysosomes have not been observed to join with phagosomes harboring single Leishmania parasites, whereas in phagosomes where the parasite has divided there is lysosome-specific fluorescence with a concomitant disappearance of lysosomes from the cytosol. In later stages, all the lysosome-specific label is found inside phagosomes whereas the phagosomal marker cadaverine strongly stains the macrophage nucleus, suggesting that Leishmania infection induces in its later stages nuclear degeneration and, possibly, apoptosis of the host cell. These results indicate that induction of macrophage apoptosis should be explored as a possible strategy used by Leishmania to prepare its egress.

Keywords: Leishmania infantum, Leishmaniasis Liposomes, Malaria, Nanomedicine, Nanotechnology, Plasmodium falciparum, Quantum dots

Fernàndez-Busquets, X., (2014). Toy kit against malaria: Magic bullets, LEGO, Trojan horses and Russian dolls Therapeutic Delivery , 5, (10), 1049-1052

Movellan, J., Urbán, P., Moles, E., de la Fuente, J. M., Sierra, T., Serrano, J. L., Fernàndez-Busquets, X., (2014). Amphiphilic dendritic derivatives as nanocarriers for the targeted delivery of antimalarial drugs Biomaterials 35, (27), 7940-7950

It can be foreseen that in a future scenario of malaria eradication, a varied armamentarium will be required, including strategies for the targeted administration of antimalarial compounds. The development of nanovectors capable of encapsulating drugs and of delivering them to Plasmodium-infected cells with high specificity and efficacy and at an affordable cost is of particular interest. With this objective, dendritic derivatives based on 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) and Pluronic® polymers have been herein explored. Four different dendritic derivatives have been tested for their capacity to encapsulate the antimalarial drugs chloroquine (CQ) and primaquine (PQ), their specific targeting to Plasmodium-infected red blood cells (pRBCs), and their antimalarial activity in vitro against the human pathogen Plasmodium falciparum and in vivo against the rodent malaria species Plasmodium yoelii. The results obtained have allowed the identification of two dendritic derivatives exhibiting specific targeting to pRBCs vs. non-infected RBCs, which reduce the in vitro IC50 of CQ and PQ by ca. 3- and 4-fold down to 4.0 nm and 1.1 μm, respectively. This work on the application of dendritic derivatives to antimalarial targeted drug delivery opens the way for the use of this new type of chemicals in future malaria eradication programs.

Keywords: Antimalarial targeted drug delivery, Dendrimers, Malaria, Nanomedicine, Plasmodium, Polymeric nanoparticles

Urbán, P., Valle-Delgado, J. J., Mauro, N., Marques, J., Manfredi, A., Rottmann, M., Ranucci, E., Ferruti, P., Fernàndez-Busquets, X., (2014). Use of poly(amidoamine) drug conjugates for the delivery of antimalarials to Plasmodium Journal of Controlled Release 177, (1), 84-95

Current malaria therapeutics demands strategies able to selectively deliver drugs to Plasmodium-infected red blood cells (pRBCs) in order to limit the appearance of parasite resistance. Here, the poly(amidoamines) AGMA1 and ISA23 have been explored for the delivery of antimalarial drugs to pRBCs. AGMA1 has antimalarial activity per se as shown by its inhibition of the in vitrogrowth of Plasmodium falciparum, with an IC50 of 13.7 μM. Fluorescence-assisted cell sorting data and confocal fluorescence microscopy and transmission electron microscopy images indicate that both polymers exhibit preferential binding to and internalization into pRBCs versus RBCs, and subcellular targeting to the parasite itself in widely diverging species such as P. falciparum and Plasmodium yoelii, infecting humans and mice, respectively. AGMA1 and ISA23 polymers with hydrodynamic radii around 7 nm show a high loading capacity for the antimalarial drugs primaquine and chloroquine, with the final conjugate containing from 14.2% to 32.9% (w/w) active principle. Intraperitoneal administration of 0.8 mg/kg chloroquine as either AGMA1 or ISA23 salts cured P. yoelii–infected mice, whereas control animals treated with twice as much free drug did not survive. These polymers combining into a single chemical structure drug carrying capacity, low unspecific toxicity, high biodegradability and selective internalization into pRBCs, but not in healthy erythrocytes for human and rodent malarias, may be regarded as promising candidates deserving to enter the antimalarial therapeutic arena.

Keywords: Malaria, Nanomedicine, Plasmodium, Polyamidoamines, Polymer-drug carriers, Targeted drug delivery

Marques, J., Moles, E., Urbán, P., Prohens, R., Busquets, M. A., Sevrin, C., Grandfils, C., Fernàndez-Busquets, X., (2014). Application of heparin as a dual agent with antimalarial and liposome targeting activities toward Plasmodium-infected red blood cells Nanomedicine: Nanotechnology, Biology, and Medicine 10, (8), 1719-1728

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.

Keywords: Heparin, Liposomes, Malaria, Plasmodium, Targeted drug delivery, Heparin, Malaria, Plasmodium, Red blood cell, Targeted drug delivery, Liposomes, 1,2 dioleoyl 3 trimethylammoniopropane, fluorescein, heparin, liposome, nanoparticle, primaquine, adsorption, animal experiment, anticoagulation, antimalarial activity, Article, binding affinity, confocal microscopy, controlled study, drug targeting, encapsulation, erythrocyte, female, fluorescence microscopy, human, human cell, in vivo study, liposomal delivery, mouse, nonhuman, Plasmodium falciparum, transmission electron microscopy

Urbán, P., Fernàndez-Busquets, X., (2014). Nanomedicine against malaria Current Medicinal Chemistry , 21, (5), 605-629

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 sp. The clinical, social and economic burden of malaria has led for the last 100 years 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. Different types of encapsulating structure, targeting molecule, and antimalarial compound will be discussed for the assembly of Trojan horse nanocapsules capable of targeting with complete specificity diseased cells and of delivering inside them their antimalarial cargo with the objective of eliminating the parasite with a single dose. Nanotechnology can also be applied to the discovery of new antimalarials through single-molecule manipulation approaches for the identification of novel drugs targeting essential molecular components of the parasite. Finally, methods for the diagnosis of malaria can benefit from nanotools applied to the design of microfluidic-based devices for the accurate identification of the parasite's strain, its precise infective load, and the relative content of the different stages of its life cycle, whose knowledge is essential for the administration of adequate therapies. The benefits and drawbacks of these nanosystems will be considered in different possible scenarios, including cost-related issues that might be hampering the development of nanotechnology-based medicines against malaria with the dubious argument that they are too expensive to be used in developing areas.

Keywords: Dendrimers, Liposomes, Malaria diagnosis, Nanobiosensors, Nanoparticles, Plasmodium, Polymers, Targeted drug delivery

Le Roux, D., Burger, P. B., Niemand, J., Grobler, A., Urbán, P., Fernàndez-Busquets, X., Barker, R. H., Serrano, A. E., I. Louw, A., Birkholtz, L. M., (2014). Novel S-adenosyl-L-methionine decarboxylase inhibitors as potent antiproliferative agents against intraerythrocytic Plasmodium falciparum parasites International Journal for Parasitology: Drugs and Drug Resistance , 4, (1), 28-36

S-adenosyl-l-methionine decarboxylase (AdoMetDC) in the polyamine biosynthesis pathway has been identified as a suitable drug target in Plasmodium falciparum parasites, which causes the most lethal form of malaria. Derivatives of an irreversible inhibitor of this enzyme, 5'-{[(Z)-4-amino-2-butenyl]methylamino}-5'-deoxyadenosine (MDL73811), have been developed with improved pharmacokinetic profiles and activity against related parasites, Trypanosoma brucei. Here, these derivatives were assayed for inhibition of AdoMetDC from P. falciparum parasites and the methylated derivative, 8-methyl-5'-{[(Z)-4-aminobut-2-enyl]methylamino}-5'-deoxyadenosine (Genz-644131) was shown to be the most active. The in vitro efficacy of Genz-644131 was markedly increased by nanoencapsulation in immunoliposomes, which specifically targeted intraerythrocytic P. falciparum parasites.

Keywords: Immunoliposomes, Plasmodium, Polyamines, S-adenosyl-l-methionine decarboxylase

Fernàndez-Busquets, X., (2013). Amyloid fibrils in neurodegenerative diseases: villains or heroes? Future Medicinal Chemistry 5, (16), 1903-1906

Fernàndez-Busquets, X., (2013). Heparin-functionalized nanocapsules: Enabling targeted delivery of antimalarial drugs Future Medicinal Chemistry 5, (7), 737-739

Pujol, A., Riera, C., Fisa, R., Molina, I., Salvador, F., Estelrich, J., Urbán, P., Fernàndez-Busquets, X., (2013). Nanomedicine for infectious diseases: Application of quantum dots encapsulated in immunoliposomes to the study of targeted drug delivery against leishmaniasis and malaria Proceedings of the 4th International Conference on Nanotechnology: Fundamentals and Applications. 4th International Conference on Nanotechnology: Fundamentals and Applications , International ASET Inc. (Ontario, Canada) , 1-8

Nanotechnological devices for therapeutic applications are massively addressed to diseases prevalent in the developed world, particularly cancer, because of the wrong assumption (for both ethical and technical reasons) that nanomedicines are too expensive and thus they can not be applied to diseases of poverty. Here we have applied quantum dots to study at the cellular level the delivery of the contents of immunoliposomes to erythrocytes infected by the malaria parasite Plasmodium falciparum, and to macrophages infected by the leishmaniasis causative agent Leishmania infantum. A number of works have reported on the encapsulation in liposomes of drugs against both diseases as a strategy to increase therapeutic efficacy and decrease unspecific toxicity. Liposome-carried drugs end up inside Plasmodium-infected red blood cells (pRBCs) and in the phagolysosome system of Leishmania-infected macrophages but some knowledge gaps still obscure subcellular events related to these processes. As a proof of concept, we have used confocal fluorescence microscopy to follow the fate in pRBCs and L. infantum-infected macrophages of quantum dots encapsulated in liposomes, and of lysosomes, Leishmania and Plasmodium parasites, nuclei, and phagosomes. Our data indicate that liposomes merge their lipid bilayers with pRBC plasma membranes but are engulfed by macrophages, where they fuse with lysosomes. Lysosomes have not been observed to join with phagosomes harboring single L. infantum parasites, whereas in phagosomes where the parasite has divided there is lysosome-specific fluorescence with a concomitant disappearance of lysosomes from the cytosol. In later stages, all the lysosome-specific label is found inside phagosomes whereas the phagosomal marker cadaverine strongly stains the macrophage nucleus, suggesting that L. infantum infection induces in its later stages nuclear degeneration and possibly, apoptosis of the host cell. These results indicate that induction of macrophage apoptosis should be explored as a possible strategy used by L. infantum to prepare its egress.

Keywords: Leishmania infantum, Leishmaniasis, Liposomes, Malaria, Nanomedicine, Nanotechnology, Plasmodium falciparum, Quantum dots

Urban, Patricia, Estelrich, Joan, Adeva, Alberto, Cortes, Alfred, Fernàndez-Busquets, X., (2011). Study of the efficacy of antimalarial drugs delivered inside targeted immunoliposomal nanovectors Nanoscale Research Letters 6, (1), 620

Paul Ehrlich's dream of a 'magic bullet' that would specifically destroy invading microbes is now a major aspect of clinical medicine. However, a century later, the implementation of this medical holy grail continues being a challenge in three main fronts: identifying the right molecular or cellular targets for a particular disease, having a drug that is effective against it, and finding a strategy for the efficient delivery of sufficient amounts of the drug in an active state exclusively to the selected targets. In a previous work, we engineered an immunoliposomal nanovector for the targeted delivery of its contents exclusively to Plasmodium falciparum-infected red blood cells [pRBCs]. In preliminary assays, the antimalarial drug chloroquine showed improved efficacy when delivered inside immunoliposomes targeted with the pRBC-specific monoclonal antibody BM1234. Because difficulties in determining the exact concentration of the drug due to its low amounts prevented an accurate estimation of the nanovector performance, here, we have developed an HPLC-based method for the precise determination of the concentrations in the liposomal preparations of chloroquine and of a second antimalarial drug, fosmidomycin. The results obtained indicate that immunoliposome encapsulation of chloroquine and fosmidomycin improves by tenfold the efficacy of antimalarial drugs. The targeting antibody used binds preferentially to pRBCs containing late maturation stages of the parasite. In accordance with this observation, the best performing immunoliposomes are those added to Plasmodium cultures having a larger number of late form-containing pRBCs. An average of five antibody molecules per liposome significantly improves in cell cultures the performance of immunoliposomes over non-functionalized liposomes as drug delivery vessels. Increasing the number of antibodies on the liposome surface correspondingly increases performance, with a reduction of 50% parasitemia achieved with immunoliposomes encapsulating 4 nM chloroquine and bearing an estimated 250 BM1234 units. The nanovector prototype described here can be a valuable platform amenable to modification and improvement with the objective of designing a nanostructure adequate to enter the preclinical pipeline as a new antimalarial therapy.

Keywords: Plasmodium falciparum, Antimalarial drug, Nanovector, Immuno-liposomes