The efficacy of self-assembled cationic antimicrobial peptide nanoparticles against Cryptococcus neoformans for the treatment of meningitis

Abstract

Cationic antimicrobial peptides have received considerable interest as new therapeutics with the potential for treatment of multiple-drug resistant infections. We recently reported that cholesterol-conjugated G₃R₆TAT (CG₃R₆TAT) formed cationic nanoparticles via self-assembly, which demonstrated strong antimicrobial activities against various types of microbes in vitro. In this study, the possibility of using these nanoparticles for treatment of Cryptococcus neoformans (yeast)-induced brain infections was studied. The antimicrobial activity of the nanoparticles was tested against 12 clinical isolates of C. neoformans in comparison with conventional antifungal agents amphotericin B and fluconazole. Minimum inhibitory concentrations (MICs) of the nanoparticles were determined to be much lower than those of fluconazole in all the isolates, but slightly higher than those of amphotericin B in some isolates. At a concentration three times higher than the MIC, the nanoparticles completely sterilized C. neoformans after 3.5 h. Cell wall disruption and release of cytoplasmic content were observed under TEM. The biodistribution studies of FITC-loaded nanoparticles in rabbits revealed that the nanoparticles were able to cross the blood-brain barrier (BBB). The efficacy of nanoparticles was further evaluated in a C. neoformans meningitis rabbit model. The nanoparticles crossed the BBB and suppressed the yeast growth in the brain tissues with similar efficiency as amphotericin B did. In addition, unlike amphotericin B, they neither caused significant damage to the liver and kidney functions nor interfered with the balance of electrolytes in the blood. CG₃R₆TAT nanoparticles can be a promising antimicrobial agent for treatment of brain infections caused by C. neoformans.

Introduction

The incidence of infections caused by the encapsulated yeast Cryptococcus neoformans has risen markedly over the past 20 years as a result of the HIV epidemic and increasing use of immunosuppressive therapies [1]. Cryptococcal meningitis is a common opportunistic infection and AIDS-defining illness especially in patients with late-stage HIV infection. It also occurs in patients with other forms of immunosuppression or even in apparently immunocompetent individuals. The use of amphotericin B induction therapy followed by consolidation therapy with fluconazole has been considered as the standard treatment for Cryptococcal meningitis [2]. Despite excellent clinical trials and evidence-based treatment guidelines, clinical response rates in HIV-infected patients are not satisfactory, with a persistently high mortality rate of 10–30%. The treatment is complicated by the poor penetration of amphotericin B across the blood–brain barrier (BBB) [3]. Approaches such as an increment in dosage, injection through the myelin sheath and liposome modification with a brain targeting peptide have been proposed to achieve therapeutic concentration of amphotericin B in the brain [4]. However, dose-dependent nephrotoxicity, infusion-related adverse effects such as fever, chills, nausea, vomiting and headache have limited the use of amphotericin B [5]. The poor penetration of antimicrobial agent into cerebrospinal fluid (CSF) is not the only factor associated with the low treatment efficiency of Cryptococcal meningitis. In addition, the increasing number of C. neoformans isolates resistant to fluconazole and amphotericin B [6], [7] represents another therapeutic challenge. Therefore, it is of increasing need to develop antimicrobial agents against C. neoformans with a new mechanism of action, which can easily cross the BBB, are broadly effective and less likely induce antimicrobial resistance.

In the search for such new antimicrobial agents, the group of antimicrobial peptides (AMPs) has attracted significant attention from academic and clinical settings during the last decade [8], [9], [10]. Compared with conventional antimicrobial agents, which are specifically active against bacteria or fungi, AMPs often exert activity against a broad spectrum of micro-organisms including bacteria, fungi, parasites, enveloped viruses and even cancer cells. These AMPs form an essential part of the “innate” arm of host resistance, serving as a first line of defense against infection. Importantly, AMPs are believed to have a mechanism of action entirely distinct from those of clinically-used antimicrobial agents. Therefore, there has been great interest in the development of AMPs for treatment of drug-resistant infections [11], [12], [13], [14]. Some AMPs are effective against C. neoformans [15], [16], but they may have limited application in the treatment of meningitis because a large dose of AMPs is needed owing to their limited ability to cross the BBB. In addition, heavy dosage of AMPs has been suspected to induce haemolysis and allergic response. Moreover, it is not easy to produce AMPs in large quantities. Therefore, there is a need to develop an alternative approach.

The HIV-1 trans-activating transcriptor (TAT) peptide is one of the most widely used molecular beacons for drug delivery [17]. Certain regions of the TAT-peptide known as protein transduction domains (PTDs), which consist of 9–16 amino acids, can pass through biological membranes via a mechanism independent of transporters and receptor-mediated endocytosis [18]. In our previous study [19], amphiphilic cholesterol-conjugated G₃R₆TAT peptide (CG₃R₆TAT), which contains TAT sequence, was designed and assembled into core/shell nanoparticles having shells anchored with TAT molecules. These nanoparticles possessed a broad spectrum of strong antimicrobial activities, much stronger than the hydrophilic peptide incapable of forming nanoparticles. The nanoparticles were also examined for in vivo activity against Staphylococcus aureus in comparison with vancomycin, a therapeutic agent that has been widely used for the prophylaxis, suppression, and treatment of S. aureus meningitis. The nanoparticles were found to be as efficient as vancomycin in treating the S. aureus meningitis in rabbits.

In this study, we examined the possibility of using these peptide nanoparticles to treat Cryptococcal meningitis. Amphotericin B, one of the most effective drugs for treating invasive fungal infection [20], [21], was employed as a positive control. Minimum inhibitory concentrations (MICs) of CG₃R₆TAT nanoparticles were first analyzed against 12 isolates of C. neoformans strains in comparison with amphotericin B and fluconazole. The efficacy of the nanoparticles in suppressing the growth of C. neoformans was then evaluated in a rabbit model of cryptococcal meningitis [22], [23], [24]. In addition, the biodistribution of FITC-loaded nanoparticles in rabbits, potential toxicity to major organs such as liver and kidney, and harmful effect on the balance of electrolytes in the blood were also investigated.