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[email protected] Current Medicinal Chemistry, 2014, 21, 605-629
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Nanomedicine Against Malaria Patricia Urbán and Xavier Fernàndez-Busquets* 1
Nanobioengineering Group, Institute for Bioengineering of Catalonia, Baldiri Reixac 10-12, Barcelona E08028, Spain; Nanomalaria Group, Barcelona Centre for International Health Research (CRESIB, Hospital Clínic-Universitat de Barcelona), Rosselló 149-153, Barcelona E08036, Spain; 3Biomolecular Interactions Group, Nanoscience and Nanotechnology Institute (IN2UB), University of Barcelona, Martí i Franquès 1, Barcelona E08028, Spain 2
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 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. INTRODUCTION Malaria is a life-threatening infectious disease which remains a major cause of morbidity and mortality in tropical and subtropical regions of the world, caused by protists of the genus Plasmodium that are transmitted to humans by infected females of certain Anopheles mosquito species. Although increased prevention and control measures have led to a reduction in mortality rates by more than 25% globally since 2000, the World Health Organization (WHO) has estimated 216 million episodes of malaria in 2010 that resulted in 655,000 deaths. People living in the poorest countries are the most vulnerable, with approximately 91% of deaths in Africa, of which 86% were children under 5 years of age [1]. Early diagnosis and treatment reduce disease, incidence and transmission of the parasite, thus contributing to shrinking the death toll. Access to diagnostic testing and treatment should be seen not only as a component of malaria control but as a fundamental right for all populations at risk. The arsenal of strategies deployed has included fighting the mosquito vector with insecticides, distributing mosquito nets, desiccating paludic areas, using an ever growing number of *Address correspondence to this author at the Nanomalaria Group, Centre Esther Koplowitz, 1st floor, CRESIB, Rosselló 149-153, Barcelona E08036, Spain; Tel: +34 93 227 5400 (ext. 4581); Fax: + 34 93 403 7181; E-mail:
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drugs against the parasite, and developing vaccination approaches, none of them being capable yet of claiming victory. This lack of success is in part due to the astonishingly refined processes evolved by the pathogen to hide, propagate, and transfer itself between hosts. MALARIA PATHOPHYSIOLOGY Five species of Plasmodium can infect and be transmitted by humans. P. falciparum, predominant in Africa, is responsible for the most deadly and severe episodes, whereas P. ovale and P. malariae induce a generally milder form of the disease that is rarely fatal. P. vivax produces less severe symptoms but it is more widespread [1], being located mainly in Asia and in South America. Severity of vivax malaria is increasing in some parts of the world and the development of drug resistance could result in an expansion of this debilitating and sometimes deadly infection [2, 3]. Finally, the zoonotic species P. knowlesi, prevalent in Southeast Asia, causes malaria in macaques but also severe infections in humans [4]. Clinical manifestations of malaria usually appear between 10 and 15 days after the mosquito bite for non-immune individuals, and consist in a wide range of symptoms similar to those of a minor systemic viral illness, including headache, nausea and cycles of fever. In endemic areas, people may develop partial immunity, allowing asymptomatic infections to occur, but complications of P. fal© 2014 Bentham Science Publishers
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ciparum malaria may lead to severe disease that could result in coma, metabolic acidosis, anemia, hypoglycemia, renal failure, acute pulmonary edema and multi-organ system failure. If not treated, severe malaria is fatal in the majority of cases, and pregnant women are at high risk of dying from complications of this form of the disease, which can be also a cause of spontaneous abortion, premature delivery, stillbirth, severe maternal anemia and low birth-weight. For P. vivax and P. ovale, clinical relapses may occur weeks to months after the first infection, even if the patient has left the malarious area. These new episodes arise from dormant liver forms known as hypnozoites, and special treatment targeted at these stages [5] is required for a complete cure. Despite the substantial impact of Plasmodium on human health, much of its basic biology is poorly understood [6]. Whilst each stage of the parasite lifecycle is the subject of intensive research, the centrality of blood stage infection to disease pathology has led to extensive effort towards understanding some of its core biological processes. LIFE CYCLE OF PLASMODIUM Malaria infection starts when a parasitized female Anopheles mosquito inoculates during a blood meal Plasmodium sporozoites, which migrate through the skin into the circulation and then to the liver. In a few minutes sporozoites invade hepatocytes, where they will develop into merozoites [7] that enter the bloodstream to invade red blood cells (RBCs) [8]. These intraerythrocytic forms grow into ring, trophozoite and schizont stages, replicating asexually to produce daughter cells that invade new erythrocytes to perpetuate the blood-stage cycle, leading to an exponential increase in parasitemia. Some parasites eventually differentiate into sexual stages, female or male gametocytes that are ingested by a mosquito from peripheral blood, and reach the insect’s midgut where micro- and macrogametocytes develop into male and female gametes through processes known as exflagellation and activation, respectively. Following fertilization the zygotes differentiate into motile and invasive ookinetes which trespass the stomach wall and attach to its outer side, where they transform into oocysts from which sporozoites are released and migrate to the mosquito salivary glands to restart the cycle at the next bite. During its intraerythrocytic phase, the parasite builds a parasitophorous vacuole inside which it proliferates [9]. The trophozoite is the metabolically most active stage, and as the pathogen matures it modifies the plasma membrane of the host RBC in order to meet its needs for growth and multiplication [10-12] and to cause adhesion of the parasitized RBC (pRBC) to the vascular endothelium. These modifications are directed at solving two problems: membrane transport processes and evasion from the immune system of the host. Although hemoglobin provides a source of protein building blocks, Plasmodium has evolved a machinery for importing nutrients from the plasma as well as for the detoxification of breakdown products resulting from hemoglobin digestion [13], which releases toxic free heme-derived compounds that are metabolized into innocuous hemozoin crystals. The properties of the pRBC membrane are modified by proteins synthesized by Plasmodium (including kinases, lipases, adhesins, proteases and chaperon-like proteins) and exported using a complex system of trafficking [14]. The identifica-
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tion of such pRBC-specific labels is of interest in the search for potential targets in targeted drug delivery strategies [15, 16]. The parasite grows and develops new organelles needed for metabolism, including a modified lysosome (digestive vacuole), and a novel system for transporting proteins beyond its own plasma membrane (Maurer’s clefts and the tubovesicular complex). Because asexual blood stages are responsible for all symptoms and pathologies of malaria, resident parasites inside pRBCs are the main target for current chemotherapeutic approaches [17]. However, the sheer numbers of pRBCs in an infected person (up to several billion) are prompting research oriented to targeting bottlenecks in the parasite life cycle represented by just a few cells in certain transmission stages [18-20], although some of these alternative cell targets pose the significant problem of their location in the mosquito. Besides a high asexual multiplication rate, there are other features of Plasmodium biology that make it a successful parasite and contribute to its survival and transmission within an ever-changing host environment. Among these are an efficient evasion of host immunity due to the unability of RBCs to process and present antigens, a high antigenic variation [21], and the redundancy in erythrocyte invasion pathways, which involve the interaction of several parasite ligands with receptors that line the RBC surface. The intraerythrocytic parasite needs to avoid passage through the spleen where resident macrophages recognize and remove RBCs with altered rheological properties. Mature stage parasites avert this spleen clearance by promoting adhesion of pRBCs to the endothelial cells of capillaries and postcapillary venules of tissues such as brain, heart, lung, kidney, placenta and small intestine. This phenomenon, known as sequestration, allows parasites to replicate while evading splenic clearance [22]. pRBCs can also adhere to noninfected RBCs giving rise to rosettes [23, 24], and they can form clumps through platelet-mediated binding to other pRBCs [25]. These events, which may lead to occlusion of the microvasculature, are thought to play a major role in the fatal outcome of severe malaria [22, 26-28]. MALARIA CONTROL, ELIMINATION AND ERADICATION Malaria control [29] is an organized attempt to prevent mortality and to reduce morbidity to a locally acceptable level using the preventive and curative tools available today in endemic countries through the progressive improvement and strengthening of local and national capabilities. Elimination of malaria [30] aims at eliminating all locally-acquired infections in a specific geographic area. Eradication, or reducing the global incidence of malaria to zero with continued measures in place to prevent re-establishment of transmission, is the long-term goal that will be achieved through progressive elimination where feasible. The malaria eradication strategy is based on three principles [31]: (i) elimination of infection and transmission in areas of low or moderate endemicity, primarily in zones at the margins of heavy transmission areas, with P. falciparum as a priority; (ii) aggressive control in the areas with high transmission; and (iii) research and development to improve existing prophylactic, diagnostic, and therapeutic tools. Progress in shrinking the geographical range of endemic malaria has been remarkable,
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and since the WHO-led Global Malaria Eradication Campaign was started in 1955 with the aim of worldwide eradication, 79 countries have eliminated malaria and the proportion of the world’s population living in endemic regions has decreased more than 50% [32]. However, efforts were gradually abandoned from 1969 to 1976 due to the realization that the objective of malaria eradication was unlikely to be easily achieved: the resistance of the vector to the insecticide dichloro-diphenyl-trichloroethane (DDT) and the evolution of drug-resistant P. falciparum strains severely impaired the WHO program [33]. In the 1990s malaria research and control strategies were accelerated [34] through the creation of several research and public health coalitions, such as the Multilateral Initiative on Malaria, the Global Fund to Fight AIDS, Tuberculosis and Malaria, the U.S. President’s Malaria Initiative and the Roll Back Malaria Partnership, which should serve as models for developing consensus on elimination. The global agenda for malaria eradication would benefit from a vaccine protecting against disease and interrupting transmission [35, 36], a work that began more than 50 years ago with some promising results such as the candidate vaccine Spf66 developed by Manuel Patarroyo [37, 38]. Nevertheless, a lack of consistency with other trials [39], the failure to prevent malaria in infants [40], and difficulties in product availability and reproducibility halted its further development [41, 42]. One of the most succesful vaccines in development is RTS,S, which is directed against the sporozoite and has attained around 40% efficacy [43], being currently in Phase III clinical trials. In the future, antigens from pre-erythrocytic, erythrocytic, sexual and mosquito stages may have to be combined into a single multi-component vaccine [35] and, if eradication is to be achieved, other malaria parasite species will need to be targeted as well, especially P.vivax. Malaria pathophysiology is too complex and the disease is too widespread to be fought with a single weapon, and it is generally accepted that to walk the path towards eradication a well-designed attack will be necessary using a combination of strategies. These include the improvement of existing approaches and the development of new ones [44], whereas drug therapy remains the mainstay of treatment and prevention [45]. Of particular interest are drugs suitable for mass administration that can be delivered in a single encounter at infrequent intervals, and that result in radical cure of all parasite stages (the so-called SERCAP: Single Encounter Radical Cure And Prophylaxis). More accurate and faster diagnosis, efficient prophylactic interventions, vector control and personal protection tools, and new, better and safer drugs administered through novel targeted delivery mechanisms will be needed to progressively eliminate malaria in each country until eradication is achieved. NANOTECHNOLOGY AND NANOMEDICINE APPLIED TO MALARIA Nanotechnology encompasses a wide range of techniques and methods for manipulating and using structures on the nanometre scale. One of the aims of nanotechnology is the development of new materials and functional devices by controlling their size and shape [46]. Nanomaterials similar in size to biologic molecules and systems may have higher
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strength, lighter weight, increased control of light spectrum, high surface area-to-volume ratio and greater chemical reactivity than their larger-scale counterparts [47]. Nanotechnology is one of the most rapidly evolving areas of science, and in the last decade it has catapulted from being a relatively small field to a global scientific and industrial enterprise. Since biological function depends heavily on elements that have nanoscale dimensions, devices at the nanoscale are small enough to interact directly with them, leading to the development of new biological technologies (e.g., singlemolecule nanobiosensors, high-sensitivity diagnosis, drug delivery systems, or imaging probes). On the other hand, nanotechnology raises concerns about the toxicity and environmental impact of novel nanomaterials. Nanotoxicology is an embryonic field and the dynamics and in vivo toxicity of nanomaterials are not well known at this time [48]. An improved understanding of the toxicological implications of nanomaterials is needed due to the enormous prospects for the application of nanotechnology in medicine. Regulatory authority guidelines must be developed quickly to ensure safe and reliable transfer of new advances in nanotechnology from laboratory to bedside. Nanomedicine is the application of nanotechnology to medicine, which is defined by the European Science Foundation [49] in the following way: “Nanomedicine uses nanosized tools for the diagnosis, prevention and treatment of disease and to gain increased understanding of the complex underlying patho-physiology of disease. The ultimate goal is improved quality-of-life”. Three main areas can be distinguished in this field: Sensors and diagnostic and imaging tools employed outside the patient. For these applications nanotechnology is used to create new analytic devices with higher sensitivity, lower cost, smaller dimensions or higher throughput. Innovative technologies and biomaterials that are used for tissue engineering, and to promote tissue repair. These applications frequently require only ex-vivo manipulations, and are aimed at the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ [50]. Drug delivery systems whose objective is to apply nanotechnology (sometimes combined with cell therapy) to the improvement of methods for the administration of pharmaceutical compounds in order to achieve more effective therapeutic effects. In the short term, nanomedicine will provide advances in drug discovery, drug delivery and analytical and diagnostic procedures. There is a growing number of marketed nanosized drug delivery systems and imaging agents, and the proliferation of bioanalytical methods for single-molecule analysis and biosensor technology based on nanotechnology represents a huge opportunity to revolutionize diagnosis in the healthcare environment. In the long term, the challenges of nanomedicine are ambitious, and include biosensors coupled to delivery systems that could combine in vivo diagnosis with a targeted, focused therapy (theranostics) [51], and imaging technologies designed with non-invasive analytical nanotools having high reproducibility, sensitivity and reliability in the simultaneous detection of several molecules for use in pre-symptom disease.
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Nanomedicine is finally making a timid appearance in the antimalarial arena in the form of diagnostic devices, nanobiosensors and, mainly, targeted drug delivery strategies, although imminent uses can also be envisaged in nanoimaging and in the application of nanoparticles to vaccination approaches to improve immune system function. MALARIA DIAGNOSIS Prompt and accurate diagnosis is part of an effective disease management [52], because if not treated malaria can quickly become life-threatening, whereas false positives increase treatment costs and drug-induced resistance, giving a wrong idea of therapeutic efficacy. Since the symptoms of malaria are nonspecific, the observation of clinical features alone might not be enough and clinical suspicion should be confirmed with a parasitological analysis. Microscopic examination of Giemsa-stained thin and/or thick blood smears remains the conventional approach for diagnosis [53]. The sensitivity of this relatively inexpensive method is excellent, allowing the detection of as few as 5 parasites per L of blood [54], and permitting also the determination of the infecting species and of the developmental stage of circulating parasites. In addition, smears provide a permanent record for quality assessment of the diagnosis. However, microscopy requires considerable expertise learned through extended training, the procedure is labor-intensive and timeconsuming, and the variability in stains and in techniques used to collect and process blood affects slide interpretation [55]. Finally, routine clinical microscopy cannot reliably detect very low parasitemias (
