Angiopep-2 modified PE-PEG based polymeric micelles for amphotericin B delivery targeted to the brain

Abstract

Amphotericin B (AmB) is a poorly water soluble antibiotic and is used to treat fungal infections of the central nervous system (CNS). However, AmB shows poor penetration into the CNS. Angiopep-2, the ligand of low-density lipoprotein receptor-related protein (LRP) present on the BBB, exhibits higher transcytosis capacity and parenchymal accumulation, which allowed us to consider the selectivity of it for receptor-mediated drug targeting to the brain. With this in mind, we prepared angiopep-2 modified PE-PEG based micellar drug delivery system loaded with the antifungal drug AmB to evaluate the efficiency of AmB accumulating into the brain. PE-PEG based micelles as nano-scaled drug carriers were investigated by incorporating AmB with high drug entrapping efficiency, improving solubilization of AmB and reducing its toxicity to mammalian cells. The AmB-incorporated angiopep-2 modified micelles showed highest efficiency in penetrating across the blood-brain barrier (BBB) than unmodified micelles and Fungizone (deoxycholate amphotericin B) in vitro and in vivo. Meanwhile, contrary to the free Rho 123, the enhancement of Rho 123-incorporated angiopep-2 modified micelles across the BBB can be explained by angiopep-2 modified polymeric micelles that have a potential to overcome the activity of efflux proteins expressed on the BBB such as P-glycoprotein. In conclusion, angiopep-2 modified polymeric micelles could be developed as a novel drug delivery system for brain targeting.

Introduction

Invasive fungal systemic infections are an important cause of morbidity and mortality in critically ill. Fungi can easily spread into the central nervous system (CNS), particularly in immunocompromized patients [1]. A vast amount of literature has been published in dealing with the CNS yeast infections such as Cryptococcus neoformans [2], Candida albicans [3] and Aspergillosis [4]. However, current therapeutic options are mostly ineffective, and mortality rates remain high.

Polyene macrolide antibiotic amphotericin B (AmB) has become a golden standard in the treatment of severe systemic fungal infections due to its unique chemotherapeutic properties (e.g., very high activity, broad antifungal spectrum, fungicidal action, and ability to overcome multidrug resistance) [5]. However, poor solubility and significant systemic toxicities, including infusion-related reactions, nephrotoxicity, hypokalemia, and hepatotoxicity, have severely limited the prevalence of conventional AmB [6], [7]. In addition, most AmB formulations, including deoxycholate amphotericin B (Fungizone) and lipid compounds of amphotericin B (such as amphotericin B lipid complex ABLC and liposomal amphotericin B AmBisome) show poor penetration into the CNS [8]. Although the liposomal form of AmB with fewer side effects has been developed in the treatment of systemic fungal infections, it needs five-fold or even more doses than conventional AmB to achieve an equivalent effect against intracerebral fungal infection [9]. In addition, amphotericin B demonstrates dose-related nephrotoxicity that limits the use of higher dosages and the duration of therapy [10]. Therefore, the AmB-incorporated polymeric micellar delivery system was established in this paper which might solve the problems such as poor solubility, low CNS-penetrating efficiency and systemic toxicities.

Amphiphilic block copolymers assemble into nano-scaled core–shell structures, micelles, which are of considerable interest for delivering drugs with poor water solubility [11]. In aqueous media, the poor water soluble drugs are entrapped into the hydrophobic cores of micelles, which increase their water solubility and bioavailability. In this study, we prepared micelles made of PEGylated phospholipids such as 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG₂₀₀₀), which is safe, biocompatible and relatively nontoxic approved by the U.S. Food and Drug Administration [12], [13]. The critical micelle concentration (CMC) of the PEGylated lipids is extremely low. This property results in some positive functions of formulated micelles such as greater solubilization of hydrophobic drugs and more thermodynamic stability upon dilution with the large volume of the blood following intravenous administration [14]. Besides solubilizing hydrophobic drugs, block copolymer micelles can also target their payloads to specific tissues through either passive or active means [15]. Block polymeric micelles such as Pluronic micelles have been previously reported to increase the delivery of agents to the brain [16]. Based on the current findings, the polymeric micelles accumulating in the CNS can be significantly enhanced via the surface modification of ligands such as antibody, peptide, lectin, saccharide, hormone, and some low-molecular-weight compounds [17]. Furthermore, PEGylation was used to prolong the circulation time of micelles in vivo, which is a prerequisite for successful drug targeting delivery.

Drug delivery systems based on the receptor-mediated mechanism have been explored to deliver drugs into the brain. Different receptors such as the transferrin receptor, the insulin receptor, and the low-density lipoprotein receptor-related protein (LRP) present on the BBB have been described as playing roles in maintaining the integrity of the BBB and brain homeostasis [18]. Among them, the low-density lipoprotein receptor-related protein (LRP) has been reported to possess the ability to mediate transport of ligands across endothelial cells of the BBB [18], [19], [20]. More than 40 different ligands of LRP ranging from lipoproteins, protease/protease inhibitor complexes, extracellular matrix proteins and viruses to growth factors and cytokines have been reported [21]. Recently, a family of peptides, named angiopeps, which are derived from the Kunitz domains of aprotinin and other human proteins known to be low-density lipoprotein receptor-related protein, have also been reported [18]. Angiopep-2 (TFFYGGSRGKRNNFKTEEY, molecular weight 2.4 kDa), a member of the angiopep family, exhibited higher transcytosis capacity and parenchymal accumulation than transferrin, lactoferrin, and avidin [22]. Moreover, angiopep-2 has been confirmed to efficiently facilitate the nanoparticles cross the BBB and accumulate in the brain [23].

In this study, we designed a formulation of 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PE-PEG) based micelles loaded with antimicrobial agent, amphotericin B, and surface-modified with angiopep-2. The efficacy of AmB-incorporated angiopep-modified, polymeric micelles entering into the CNS was evaluated in the aspects of internalization by brain capillary endothelial cells (BCECs) in vitro and the brain tissue distribution in vivo. The cytotoxicity and hemolysis between the conventional AmB and AmB-incorporated micelles were also investigated.