Th-1 biased immunomodulation and synergistic antileishmanial activity of stable cationic lipid–polymer hybrid nanoparticle: Biodistribution and toxicity assessment of encapsulated amphotericin B

Highlights

• Macrophage targeted stearylamine lipid–polymer hybrid nanoparticles are discussed.

• Encapsulated amphotericin B mediated toxicity gets reduced.

• Amphotericin B and stearylamine show synergistic antileishmanial effect.

• Th-1 biased immunomodulatory effect enhanced microbicidal activity of macrophages.

• Promising alternative to problematic commercial Amphotericin B formulations.

Abstract

To address issues related to Amphotericin B (AmpB) clinical applications, we developed macrophage targeted cationic stearylamine lipid–polymer hybrid nanoparticles (LPNPs) with complementary characteristics of both polymeric nanoparticles and liposomes, for enhancement of therapeutic efficacy and diminishing toxic effect of encapsulated AmpB. The LPNPs (size 198.3 ± 3.52 nm, PDI 0.135 ± 0.03, zeta potential +31.6 ± 1.91 mV) provide core-shell type structure which has the ability to encapsulate amphiphilic AmpB in higher amount (Encapsulation efficiency 96.1 ± 2.01%), sustain drug release and stabilize formulation tremendously. Attenuated erythrocytes and J774A.1 toxicity of LPNPs demonstrated safe applicability for parenteral administration. Elevated macrophage uptake of LPNPs, rapid plasma clearance and higher drug allocation in macrophage abundant liver and spleen illustrated admirable antileishmanial efficacy of AmpB-LPNPs in vitro (IC₅₀, 0.16 ± 0.04 μg AmpB/ml) and in vivo (89.41 ± 3.58% parasite inhibition) against visceral leishmaniasis models. Augmentation in antileishmanial activity due to Th-1 biased immune-alteration mediated by drug-free LPNPs which elevated microbicidal mediators of macrophages. Moreover, minimal distribution to kidney tissues and low level of nephrotoxicity markers (creatinine and BUN) demonstrated the safety profile of AmpB-LPNPs. Conclusively, reliable safety and macrophage directed therapeutic performance of AmpB-LPNPs suggest it as promising alternative to commercial AmpB-formulations for the eradication of intra-macrophage diseases.

Introduction

Mononuclear phagocytes (macrophages/dendritic cells) are the professional antigen presenting cells (APCs) of the immune system that play central role in bridging innate and adaptive immune responses to act against microbial pathogens in a coordinated manner. As indicated by designation, primary role of these cells is phagocytosis causing destruction of ingested pathogen. Internalization within APCs is mediated through recognition of pathogen-associated molecular patterns (PAMPs) particular for every pathogen, by cellular pattern recognition receptors (PRRs) such as stearylamine receptors, toll-like receptors, lectin-receptors, CD14 or various classes of scavenger receptors, located on APCs [1], [2]. Engagement of PRRs by their PAMPs activates APCs, stimulating cytokine secretion that regulates the adaptive immune response, and promotes upregulation of co-stimulatory molecules in order to improve antigen presentation to T cells.

Mononuclear phagocytic system (MPS) plays a pivotal role in the pathophysiology of certain specific diseases including chronic obstructive pulmonary disease (COPD), asthma [3] and cancer [4], [5] and in parasitic infections including leishmaniasis [6], tuberculosis [7] and human immunodeficiency virus (HIV) infection [8] and is a major target for future therapy of mentioned MPS mediated diseases. As a result, drug delivery systems that can target APCs are crucial and could usher in an unorthodox but highly effective therapeutic paradigm for a wide range of diseases.

Cure of intra-APCs parasitic diseases including leishmaniasis, even during chemotherapy, depends upon the development of an effective immune response that activates APCs to kill the intracellular parasites. APCs do this by increasing production of proinflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-12 and mediators such as nitric oxide (NO) and reactive oxygen intermediates (ROI) [9], [10], [11]. However, this expected immune response is suppressed by the infection which interferes in the mandatory signaling between APCs and T cells, such as IL-12 production and MHC presentation [12], [13]. Hence, synergistic chemotherapy with prospective immunomodulatory role may provide a strengthened therapeutic strategy capable of shortening the duration of treatment as well as ensuring the persistent therapeutic effect of drug.

Amphotericin B (AmpB) has long been recognized as the most effective drug for the treatment of mycosis as well as protozoan infections, viz. leishmaniasis, with great commercial success. However, dose-limiting adverse events, particularly renal insufficiency and hematologic toxicity have somewhat dented its clinical relevance [14]. Investigation toward reduction of free AmpB and conventional AmpB deoxycholate (dAmpB) mediated toxicity, has led to development of liposomal formulation (LAmpB); however, its poor scale-up, stability and cost concern [15] still leave a lot to be desired. Consequently, efforts are needed to develop a highly stable and cost effective delivery tool that can subdue toxicity of drug by delivering it directly to parasite residence site (APCs). In this respect, incorporation of PRR specific ligand or immunomodulatory moieties including proteins, antibodies, polysaccharides, glycolipids, glycoproteins and lectins, into polymeric nanoparticles could be a beneficial strategy for improving efficacy of drug targeting and eliminating undesirable side actions. The comparative higher stability profile of polymeric nanoparticles accompanied by the low cost of its components provides economic relevance to the delivery system raising the affordability quotient.

Accordingly in the present study, we architected lipid–polymer hybrid nanoparticles (LPNPs) by utilizing poly (d,l-lactide-co-glycolide) (PLGA) as a biocompatible polymer [16], while stearylamine (Sta) as lipid was selected due to its cationic nature, biocompatibility, benign antiprotozoan activity and immuno-potentiation strength [17]. Since stearylamine also acts as a ligand for stearylamine PRR present on macrophage surface and phosphatidylserine biomarker of Leishmania parasite ligand [17], its presence on the surface of nanoparticles may serve as targeting moiety as well. Herein, we demonstrate a practical drug delivery system for better management of visceral leishmaniasis.