Development and evaluation of macrophage targeted multidrug therapy against visceral leishmaniasis

Visceral leishmaniasis (VL) also known as kala-azar caused by “Leishmania donovani” and transmitted by the sandfly, “Phlebotomus argentipes” is the most severe form of leishmaniasis and is almost fatal, if left untreated. It results from infection of the macrophages of the liver, spleen and bone marrow. The symptoms of conventional disease are fever, weight loss, hepatosplenomegaly and pancytopenia. It occurs in more than 80 countries in Africa, Asia, Southern Europe and South America. Approximately 500,000 new cases of VL occur worldwide each year. India alone may contribute as much as 40-50% of these with 90% occur in the state of Bihar (1). Despite the accessibility of several drugs, currently existing therapies for VL are very toxic and also started showing emergence of drug-resistant parasitic strains. For instance, pentavalent antimonials (sodium stibogluconate) and pentamidine were potent drugs used against VL; but there is emerging evidence that the rates of response are declining due to the appearance of resistance, relapses due to the emergence of several unresponsive strains of the leishmanial parasite and toxicity (hypotension, reversible hypoglycemia, and nephrotoxicity) (2). Miltefosine is a relatively new anti-VL drug which holds significant potential in treating VL along with other first-line therapeutics (3); however, primary mechanism is quite uncertain, and possible impairment of lipid metabolism occurs. Despite all research initiatives and preclinical studies, effective therapy for VL still remains challenge holding direct negative implications with respect to better drug targeting and overcoming drug resistance (3). Amphotericin B (AmB) is used as a prototype leishmanicidal drug due to its excellent efficacy and its use results in fewer treatment failures and relapses (4, 5). However, the important side effects, mainly nephrotoxicity, produced by this drug at therapeutic doses have often led to its rejection as a first-choice treatment. Counter intuitively, doxorubicin (DOX), an anthracycline antibiotic widely used anticancer agents (6), was reported to be equally effective (in comparison to amphotericin) as an anti-VL agent based on the studies conducted by Sett et al. and other researchers (7). Although highly potent, activity of DOX has been restricted by its fatal toxicity, cost-effectiveness, dose limited cardiotoxicity and myelosuppression (8, 9). The problem requires that the drug should be targeted to the macrophages in such a way that the interaction of the free drug Abstract In this study, we fabricated PCL-nanoparticles by encapsulating dual drugs as amphotericin B and doxorubicin via doubleemulsion solvent evaporation method also incorporated with ligand-lectin for targeting the infested macrophage cells and prove importance against VL. Different independent processing parameters were assessed systematically to enhance the incorporation of the dual agents with different properties (AmB and DOX, hydrophobic & hydrophilic molecule, respectively) into PCL-NPs and control particle size. Approaches investigated for the enhancement of drug entrapment efficiencies and smaller particle size included the influence of the drug content, polymer content, sonication time etc. The mean particle size and zeta potential of PCLNPs were 236.7 ± 0.04 nm in diameter and -9.11 ± 3.46 mV, respectively. The entrapment efficiencies of AmB and DOX were 82.1 ± 1.39 and 75.20 ± 0.14 %, respectively. Antileishmanial activities of the formulations and various combination approaches were assessed using macrophage-specific ligand-lectin. The prepared plain and lectin coated PCL-NPs based systems showed remarkable potential for passive and active intra macrophage targeting, respectively and the approach could be a successful alternative to the currently available drug regimens against VL. Multidrug resistance can be improved by combination delivery of encapsulated anti VL drugs. Thus, the co-encapsulation of AmB and DOX should reduce side effects of both drugs while increasing efficacy.