Activity and in vivo tracking of Amphotericin B loaded PLGA nanoparticles

doi:10.1016/j.ejmech.2015.03.022

European Journal of Medicinal Chemistry

Volume 95, 5 May 2015, Pages 267-276

https://doi.org/10.1016/j.ejmech.2015.03.022 Get rights and content

Highlights

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Amphotericin B is a widely used antifungal, but toxicity limits its use.
•
NANO-D-AMB was proposed to optimize efficacy and reduce toxicity of this drug.
•
NANO-D-AMB had similar antifungal efficacy than AMBISOME^®.
•
NANO-D-AMB had lower toxicity compared to D-AMB in vivo and in vitro.
•
NANO-D-AMB improves drug delivery and might be a potential alternative to D-AMB.

Abstract

The development of biocompatible polymeric nanoparticles has become an important strategy for optimizing the therapeutic efficacy of many classical drugs, as it may expand their activities, reduce their toxicity, increase their bioactivity and improve biodistribution. In this study, nanoparticles of Amphotericin B entrapped within poly (lactic-co-glycolic) acid and incorporated with dimercaptosuccinic acid (NANO-D-AMB) as a target molecule were evaluated for their physic-chemical characteristics, pharmacokinetics, biocompatibility and antifungal activity. We found high plasma concentrations of Amphotericin B upon treatment with NANO-D-AMB and a high uptake of nanoparticles in the lungs, liver and spleen. NANO-D-AMB exhibited antifungal efficacy against Paracoccidioides brasiliensis and induced much lower cytotoxicity levels compared to D-AMB formulation in vivo and in vitro. Together, these results confirm that NANO-D-AMB improves Amphotericin B delivery and suggest this delivery system as a potential alternative to the use of Amphotericin B sodium deoxycholate.

Graphical abstract

Biodistribution scintigraphic images of nanoparticles labeled with the radioisotope technetium 99: amphotericin loaded nanoparticles (99mTc-NANO-D-AMB) or free DMSA after 1 and 8 h of intravenously injection.

Introduction

Amphotericin B is a natural polyene antifungal commonly used in therapy against severe systemic fungal infections [1]. The administration of this drug is done by intravenous injection, which is quite inconvenient for long periods because of the need for continuous and prolonged venous access, in addition to the adverse effects and toxicity related to its use [1], [2]. Severe adverse toxic side effects are associated with the use of Amphotericin B sodium deoxycholate (D-AMB), mainly renal failure. Alternative nanostructure-based formulations, such as lipid formulations and polymeric nanoparticles, have demonstrated antifungal efficacy and lower toxicity, decrease in the active principle dose and increase in the accessibility of the drug to organs and targeted tissue [1], [3].

Lipid-based preparations for Amphotericin B, such as liposomal Amphotericin B (AMBISOME^®) considerably reduced the toxicity caused by this drug [4]. Recently, polymeric formulations composed of poly (lactic-co-glycolic) acid (PLGA) have also been proposed as a promising alternative to circumvent the adverse effects induced by treatment with this antifungal [5], [6], [7], [8], [9], [10]. This strategy is also appropriate for target-delivery of the drug to specific organs in the body [3].

Previous data from our group [3] proposed a new formulation for D-AMB entrapped within PLGA and dimercaptosuccinic acid (DMSA) nanoparticles (NANO-D-AMB). It was demonstrated that NANO-D-AMB had the same antifungal effectiveness as D-AMB in vivo, with the advantage of being administered each three days (instead of daily), with sustained release of Amphotericin B and no increase in the toxic effects of the treatment, thus allowing a reduction in the number of injections [3].

NANO-D-AMB nanoparticles were evaluated in vivo against paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America, characterized as a lung chronic granulomatous infection caused by the thermo-dimorphic fungus from Paracoccidioides genre [11]. D-AMB is the therapeutic option for the severe forms of PCM, and it is used as the first choice in intravenous therapy until the remission of the disease, in which the patient should be transferred to the maintenance phase with the use of oral drugs, such as trimetrophim-sulphametoxazol and azoles [12]. Since the infection is located mainly in the lungs, it was useful to evaluate the polymeric Amphotericin B formulation associated with dimercaptosuccinic acid, which has a preferential tropism to the lungs [13].

These previous results prompted questions about physic-chemical characteristics of the NANO-D-AMB nanoparticle and its biosafety and biodistribution. The present study reports the physic-chemical characterization of NANO-D-AMB and its biocompatibility in vivo and in vitro, apart from a comparison of antifungal effectiveness in vivo with D-AMB and AMBISOME^®.

Section snippets

Material and methods

Amphotericin B sodium deoxycholate (Sigma, St Louis, MO, USA) containing 45% of pure Amphotericin B and 35% of sodium deoxycholate was used as treatment and to prepare nanoparticles. The polylactic acid (PLA), polyglycolic acid (PGA) and DMSA used to prepare the nanoparticles were also purchased from Sigma (St Louis, MO, USA). AMBISOME^® (Gilead, USA), the liposomal formulation of Amphotericin B, was the therapeutic agent used to compare the in vivo antifungal activity, and Anforicin B^®

Physicochemical and morphological characterization

The TEM analysis of the nanoparticles demonstrated a uniform spherical shape, with a variation in size distribution, including both isolated and agglomerated particles (Fig. 1A–D). The SEM analysis of the nanoparticles (Fig. 1E–H) supported TEM data, evidencing a smooth nanoparticle surface and slight aggregation.

Average diameter (D), zeta potential (ZP) and polydispersity index (PI) of NANO-D-AMB and NANO-PLGA were measured by the range of time of 21 days and were summarize in Table 1.

In vitro release of Amphotericin B from nanoparticles

The

Discussion

The data presented in this study reinforce the findings observed by Amaral et al. [3], in which Amphotericin B encapsulated within PLGA and DMSA nanoparticles (NANO-D-AMB) was used to treat paracoccidioidomycosis, presenting antifungal activity and preventing nephrotoxic effects in mice. In this previous work, in vivo assays also showed that NANO-D-AMB was able to reduce the fungal burden in mice lungs at the same levels as D-AMB, with the advantage of allowing a reduction in the number of

Conflict of interest

There is no conflict of interest.

Acknowledgments

The authors wish to thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) (554349/2008-6 e 559157/2008-8) and FINEP (Financiadora de Estudos e Projetos) for their financial support. The authors also thank the Laboratory of Clinical Analysis from the University Hospital of Brasília (Brasília/DF).

References (50)

R. Laniado-Laborín et al.
Amphotericin B: side effects and toxicity
Rev. Iberoam. Micol.
(2009)
J. Khandare et al.
Polymer–drug conjugates: progress in polymeric prodrugs
Prog. Polym. Sci.
(2006)
M.S. Ferreira
Paracoccidioidomycosis
Paediatr. Respir. Rev.
(2009)
M.P. Garcia et al.
Morphological analysis of mouse lungs after treatment with magnetite-based magnetic fluid stabilized with DMSA
J. Magn. Magn. Mater.
(2005)
M. Nahar et al.
Development, characterization, and toxicity evaluation of amphotericin B-loaded gelatin nanoparticles
Nanomedicine
(2008)
L.H. Wang et al.
High-performance liquid chromatographic analysis of amphotericin B in plasma, blood, urine and tissues for pharmacokinetic and tissue distribution studies
J. Chromatogr.
(1992)
I. Echevarría et al.
High-performance liquid chromatographic determination of amphotericin B in plasma and tissue. Application to pharmacokinetic and tissue distribution studies in rats
J. Chromatogr. A
(1998)
P.O. Gubbins et al.
Rapid and sensitive high performance liquid chromatographic method for the determination of itraconazole and its hydroxy-metabolite in human serum
J. Pharm. Biomed. Anal.
(1998)
E. Tessier et al.
High-performance liquid chromatographic mass spectrometric method for the determination of ursodeoxycholic acid and its glycine and taurine conjugates in human plasma
J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
(2003)
A.F. Marques et al.
Additive effect of P10 immunization and chemotherapy in anergic mice challenged intratracheally with virulent yeasts of Paracoccidioides brasiliensis
Microbes Infect.
(2008)

T. Ehrenfreund-Kleinman et al.

Synthesis and characterization of novel water soluble amphotericin B-arabinogalactan conjugates

Biomaterials

(2002)

D. Anderson et al.

Cyclophosphamide: review of its mutagenicity for an assessment of potential germ cell risks

Mutat. Res.

(1995)

A.M. Ribeiro et al.

HSP65 DNA as therapeutic strategy to treat experimental paracoccidioidomycosis

Vaccine

(2010)

A.M. Ribeiro et al.

DNAhsp65 vaccination induces protection in mice against Paracoccidioides brasiliensis infection

Vaccine

(2009)

H.J. Jeon et al.

Effect of solvent on the preparation of surfactant-free poly(DL-lactide-co-glycolide) nanoparticles and norfloxacin release characteristics

Int. J. Pharm.

(2000)

T. Govender et al.

PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug

J. Control Release

(1999)

M. Kleinberg

What is the current and future status of conventional amphotericin B?

Int. J. Antimicrob. Agents

(2006)

K. Avgoustakis et al.

Effect of copolymer composition on the physicochemical characteristics, in vitro stability, and biodistribution of PLGA-mPEG nanoparticles

Int. J. Pharm.

(2003)

B. Semete et al.

In vivo evaluation of the biodistribution and safety of PLGA nanoparticles as drug delivery systems

Nanomedicine

(2010)

D. Ellis

Amphotericin B: spectrum and resistance

J. Antimicrob. Chemother.

(2002)

A.C. Amaral et al.

Amphotericin B in poly(lactic-co-glycolic acid) (PLGA) and dimercaptosuccinic acid (DMSA) nanoparticles against paracoccidioidomycosis

J. Antimicrob. Chemother.

(2009)

S.A. Costa Lima et al.

Characterization and evaluation of BNIPDaoct-loaded PLGA nanoparticles for visceral leishmaniasis: in vitro and in vivo studies

Nanomedicine (Lond)

(2012)

J.L. Italia et al.

Peroral amphotericin B polymer nanoparticles lead to comparable or superior in vivo antifungal activity to that of intravenous Ambisome^® or Fungizone^™

PLoS One

(October 2011)

J.L. Italia et al.

Biodegradable nanoparticles improve oral bioavailability of amphotericin B and show reduced nephrotoxicity compared to intravenous Fungizone

Pharm. Res.

(2009)

K.K. Jain

Applications of nanobiotechnology in clinical diagnostics

Clin. Chem.

(2009)

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¹: These authors equally contributed to this work.

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Original articleActivity and in vivo tracking of Amphotericin B loaded PLGA nanoparticles

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Material and methods

Physicochemical and morphological characterization

In vitro release of Amphotericin B from nanoparticles

Discussion

Conflict of interest

Acknowledgments

Rev. Iberoam. Micol.

Prog. Polym. Sci.

Paediatr. Respir. Rev.

J. Magn. Magn. Mater.

Nanomedicine

J. Chromatogr.

J. Chromatogr. A

J. Pharm. Biomed. Anal.

J. Chromatogr. B Anal. Technol. Biomed. Life Sci.

Microbes Infect.

Biomaterials

Mutat. Res.

Vaccine

Vaccine

Int. J. Pharm.

J. Control Release

Int. J. Antimicrob. Agents

Int. J. Pharm.

Nanomedicine

Amphotericin B: spectrum and resistance

J. Antimicrob. Chemother.

Amphotericin B in poly(lactic-co-glycolic acid) (PLGA) and dimercaptosuccinic acid (DMSA) nanoparticles against paracoccidioidomycosis

J. Antimicrob. Chemother.

Characterization and evaluation of BNIPDaoct-loaded PLGA nanoparticles for visceral leishmaniasis: in vitro and in vivo studies

Nanomedicine (Lond)

Peroral amphotericin B polymer nanoparticles lead to comparable or superior in vivo antifungal activity to that of intravenous Ambisome® or Fungizone™

PLoS One

Biodegradable nanoparticles improve oral bioavailability of amphotericin B and show reduced nephrotoxicity compared to intravenous Fungizone

Pharm. Res.

Applications of nanobiotechnology in clinical diagnostics

Clin. Chem.

Original article
Activity and in vivo tracking of Amphotericin B loaded PLGA nanoparticles

Peroral amphotericin B polymer nanoparticles lead to comparable or superior in vivo antifungal activity to that of intravenous Ambisome^® or Fungizone^™