Original Article
Novel biodegradable poly(gamma-glutamic acid)–amphotericin B complexes show promise as improved amphotericin B formulations

https://doi.org/10.1016/j.nano.2017.02.003Get rights and content

Abstract

Commercially available amphotericin B (AmB) formulations are limited by cytotoxicities, lower efficacies, shelf-life related issues and high production costs. In this study, AmB complexes based on poly(gamma-glutamic acid) (PGGA) were prepared and evaluated for their efficacies against AmB-deoxycholate (Fungizone®) and liposomal AmB (AmBisome®). Physical characterizations showed that AmB/PGGA complexes are nanoscopic (20-40 nm) with a negative zeta potential (−45.5 to −51.0 mV), water-soluble, stable in solution (up to 4 weeks, at 4 °C and 25 °C), and have a high drug loading (up to 35% w/w). In vitro, AmB/PGGA complexes exhibited a more favorable cytotoxicity profile than Fungizone® but comparable to AmBisome®, with respect to the hemolytic activity and the modulation of pro-inflammatory cytokines (TNF-α and IL-1ß). In-vivo, AmB/PGGA complexes were significantly more efficacious than both Fungizone® and AmBisome® against experimental murine candidiasis. These results provide strong evidence that AmB/PGGA complexes display better efficacy and safety features than the currently approved AmB products.

Section snippets

Degradation of high molecular weight PGGA

In a typical alkaline hydrolysis reaction, PGGA of the free acid form (5 g, 34.45 mmol; 1500 kDa, Natto Biosciences) was dissolved in aqueous sodium bicarbonate (50 mL, 67.60 mM). The solution was heated to 90 °C and NaOH (1.38 g, 34.45 mmol) was added. The solution was maintained at 90 °C for 6 h, and subsequently allowed to cool to room temperature. The pH of the solution was adjusted to 7.0 with HCl, and subsequently treated with Amberlite resin (50 g, IR-120 H+ type, 50 mesh,

Molecular weight determination by gel permeation chromatography (GPC)

The results of PGGA degradation are shown in Table 1. For the determination of MW of the PGGA, the specific refractive index (dn/dc) of the polymer was obtained from a plot of refractive index (IR) detector peak area versus the PGGA concentration (0-10 mg/mL) at a fixed volume (1 mL); the slope of which corresponded to the dn/dc value (0.0939) (Figure S1). The polymer MW was calculated using the Rayleigh equation:Rθθ0KCM

Where R, K, C and M are the intensity of the scattered light, the optical

Discussion

Our goal was to develop an alternative AmB formulation that offers the best attributes of the approved AmB formulations, namely the high efficacy and low cost of the AmB-deoxycholate, along with improved safety profiles of the lipid based AmB formulations. Using the naturally-occurring and biodegradable polymer PGGA, we were able to synthesize non-covalently associated AmB/PGGA complexes of varying polymer molecular weights (55, 65 and 110 kDa). The preparation of the AmB/PGGA complexes

References (60)

  • Z.X. Liao et al.

    Enhancement of efficiencies of the cellular uptake and gene silencing of chitosan/siRNA complexes via the inclusion of a negatively charged poly(γ-glutamic acid)

    Biomaterials

    (2010)
  • S.F. Peng et al.

    Mechanisms of cellular uptake and intracellular trafficking with chitosan/DNA/poly(γ-glutamic acid) complexes as a gene delivery vector

    Biomaterials

    (2011)
  • J. Fu et al.

    Macrophage mediated biomimetic delivery system for the treatment of lung metastasis of breast cancer

    J Control Release

    (2015)
  • J. McMillan et al.

    Cell delivery of therapeutic nanoparticles

    Prog Mol Biol Transl Sci

    (2011)
  • H.A. Gallis et al.

    Amphotericin B: 30 years of clinical experience

    Reviews Infect Dis

    (1990)
  • G. Deray

    Amphotericin B nephrotoxicity

    J Antimicrob Chemother

    (2002)
  • G. Inselmann et al.

    Influence of amphotericin B treatment duration of hepatic microsomal enzyme function in rats

    Pharmacology

    (1997)
  • M.W. Pound et al.

    Overview of treatment options for invasive fungal infections

    Med Mycol

    (2011)
  • AmBisome®, summary of product characteristics

  • Fungizone®, summary of product characteristics

  • S.B. Girois et al.

    Adverse effects of antifungal therapies in invasive fungal infections: review and meta-analysis

    Eur J Clin Microbiol Infect Dis

    (2006)
  • F. Zhao et al.

    Cellular uptake, intracellular trafficking, and cytotoxicity of nanomaterials

    Small

    (2011)
  • S. Gupta et al.

    Development and characterization of amphotericin B bearing emulsomes for passive and active macrophage targeting

    J Drug Target

    (2007)
  • M.P. Melancon et al.

    Multifunctional synthetic poly(L-glutamic acid)-based cancer therapeutic and imaging agents

    Mol Imaging

    (2011)
  • J.-W. Yoo et al.

    Bio-inspired, bioengineered and biomimetic drug delivery carriers

    Nat Rev Drug Discov

    (2011)
  • V.P. Torchilin

    Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery

    Nat Rev Drug Discov

    (2014)
  • P.R. Veerareddy et al.

    Antileishmanial activity, pharmacokinetics and tissue distribution studies of mannose grafted amphotericin B lipid nanospheres

    J Drug Target

    (2009)
  • Q. Zia et al.

    Novel drug delivery systems for antifungal compounds, pgs 485–528

  • J. Jee et al.

    Encapsulation and release of amphotericin B from an ABC triblock fluorous copolymer

    Pharm Res

    (2012)
  • H. Chen et al.

    Fast release of lipophilic agents from circulating PEG-PDLLA micelles revealed by in vivo forster resonance energy transfer imaging

    Langmuir

    (2008)
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    Funding: We would like to thank Natto Biosciences for the kind donation of poly(gamma-glutamic acid) for this research.

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