Pharmaceutical nanotechnology
Drug delivery by polymeric nanoparticles induces autophagy in macrophages

https://doi.org/10.1016/j.ijpharm.2011.11.020Get rights and content

Abstract

Drug delivery nanosystems are currently used in human therapy. In preliminary studies we have observed that Eudragit® RS nanoparticles, prepared by nanoprecipitation or double emulsion techniques, are cytotoxic for NR8383 rat macrophages. In this study, we expand our previous analysis and suggest that unloaded Eudragit® RS nanoparticles prepared by nanoprecipitation (NP/ERS) may induce important morphological and biochemical cellular modifications leading to cellular death. In NR8383 rat macrophages cell line exposed to doses varying from 15 to 100 μg/mL, NP/ERS nanoparticles are internalized inside the cells, reach the mitochondria and alter the structure of these organelles. In addition, the exposure to nanoparticles induces cellular autophagy as demonstrated by electron microscopy analysis, microchip array, qRT-PCR and Western blot assays. Although toxicity of nanoparticles has already been evidenced, it is the first time that results show clearly that the toxicity of polymeric nanovectors may be related to an activation of autophagy.

Introduction

Nano-drug delivery systems (NDDS) based on polymeric biomaterials have received considerable interest as drug delivery vehicles, and as nonviral gene delivery systems (Unger et al., 2007). Nanoparticles used as drug delivery vehicles are generally <500 nm in at least one dimension, and consist of different biodegradable or non biodegradable materials such as natural or synthetic polymers, lipids or metals (Hoffart et al., 2006, Suri et al., 2007). Despite their wide use, their toxicity is seldom evaluated and the studies are limited, in most cases, to the effect of loaded nanoparticles. On the contrary, the toxicity of the unloaded nanoparticles is not investigated and the mechanisms through which they might interfere with the cellular metabolism are largely unknown. Among the numerous available nanoparticles, those prepared from Eudragit® RS (ERS), a non-biodegradable positively charged copolymer, licensed for clinical use by the major health authorities of Europe, Japan and USA (Hoffart et al., 2006), are efficient NDDS. ERS nanoparticles prepared by nanoprecipitation (NP) or by double emulsion (DE) techniques containing ibuprofen (Pignatello et al., 2002), cyclosporin (Pignatello et al., 2002), indomethacin (Bhardwaj et al., 2010), melatonin (Schaffazick et al., 2008), DNA plasmid (Gargouri et al., 2009) and low molecular weight heparin (Jiao et al., 2002) (LMWH) have been obtained and suggested to be used for the treatment of different pathological conditions. A previous work from our laboratory has already shown, by using MTT and Trypan blue exclusion tests, that unloaded NP/ERS nanoparticles, at a final concentrations ranging from 25 to 400 μg/mL, have cytotoxic effects on the rat macrophage cell line NR8383 (Eidi et al., 2010). Additionally, other studies performed on ERS nanoparticles toxicity are scarce and often they are limited to cell survival (Gargouri et al., 2009, Lamprecht et al., 2006). Taking into consideration that (i) 44% of the total nanoparticles present in a formulation is empty (Eidi et al., 2010); (ii) as demonstrated in rabbits, nanoparticles reach the blood stream after oral administration (Hoffart et al., 2006) and that (iii) macrophages are among the first cells they interact with, we extended our study on the effect of NP/ERS on NR8383 rat macrophages, which evolve in fully activated macrophages (Nguea et al., 2008), with the aim to better elucidate the molecular mechanisms that may be responsible of the observed cytotoxicity and so to further address the safety issues surrounding nanoparticles use.

Section snippets

Materials

Eudragit® RS PO (ERS; MW = 150,000 Da) was a gift of Evonik polymers (Darmstadt, Germany). CAS number: 33434-24-1, chemical/IUPAC name: poly(ethyl acrylate-co-methyl methacrylate-co trimethylammonioethyl methacrylate chloride) 1:2:0.1. INCI name: Acrylates/Ammonium Methacrylate Copolymer, molecular weight: 32 g/mol. Eudragit® RS PO was described in the following monographs: (i) European pharmacopoeia: Ammonio Methacrylate Copolymer, Type B; (ii) USA pharmacopoeia: Ammonio Methacrylate Copolymer,

Nanoparticle characterization

NP/ERS nanoparticles were positively charged (+40.6 ± 5 mV) owing to the quaternary ammonium groups of the polycationic ERS polymer. In addition, these nanoparticles had a mean nominal diameter value of 54.2 ± 6 nm and a weak polydispersity index (0.5 ± 0.04). Particle size results obtained by Zetasizer™ were comparable to those measured by SEM analysis (Fig. 1).

TEM observations

Uptake of particles occurred in NR8383 macrophages upon incubation for 2 h with nanoparticles at a final concentration of 15 and 25 μg/mL. The

NP/ERS uptake by NR8383 macrophages and mitochondria targeting

It has been reported that endocytosis pathway plays a fundamental role in a wide range of cellular function; in particular it affects the functions of mitochondria (Polo and Di Fiore, 2006). Uptake of particles by endocytosis depends primarily on their size and ionic charge (Xia et al., 2006). It has been documented that cationic and small diameter nanoparticles, such as 60 nm NH2-labeled nanospheres, are internalized into the cells and gain access to cellular organelles and may damage

Conclusion

Nanotechnology has attracted increasing interest among almost all fields of research. Notable improvements in our knowledge about metallic nano-objects or carbon nanotubes toxicity and their influence on the expression of genes and the impairment of the oxidant/antioxidant cellular balance have been obtained. Nevertheless, data on the toxicity of drug delivery nanoparticles remain extremely scarce. To the best of our knowledge, this article is the first to report that polymeric nanoparticles

Acknowledgements

The authors thank Dr. Lucia Marcocci for her help and support in reviewing the manuscript. Authors would like to acknowledge Lu Zhang, Kevin Dalleau, Bouchra Mouaraki, Christine Manencq and Ramia Safar for their kind help in microarray and PCR experiments.

References (52)

  • R. Pignatello et al.

    Eudragit RS100 nanosuspensions for the ophthalmic controlled delivery of ibuprofen

    Eur. J. Pharm. Sci.

    (2002)
  • S. Polo et al.

    Endocytosis conducts the cell signaling orchestra

    Cell

    (2006)
  • S.R. Schaffazick et al.

    Incorporation in polymeric nanocapsules improves the antioxidant effect of melatonin against lipid peroxidation in mice brain and liver

    Eur. J. Pharm. Biopharm.

    (2008)
  • F. Unger et al.

    Branched polyesters based on poly[vinyl-3-(dialkylamino)alkylcarbamate-co-vinyl acetate-co-vinyl alcohol]-graftpoly(d,l-lactide-co-glycolide): effects of polymer structure on cytotoxicity

    Biomaterials

    (2007)
  • D. Wallach et al.

    Cell death induction by receptors of the TNF family: towards a molecular understanding

    FEBS Lett.

    (1997)
  • H. Wang et al.

    Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader

    Free Radic. Biol. Med.

    (1999)
  • Z. Yang et al.

    Pharmacological and toxicological target organelles and safe use of single-walled carbon nanotubes as drug carriers in treating Alzheimer disease

    Nanomedicine

    (2010)
  • K. Asanuma et al.

    MAP-LC3, a promising autophagosomal marker, is processed during the differentiation and recovery of podocytes from PAN nephrosis

    FASEB J.

    (2003)
  • P. Bhardwaj et al.

    Formulation and in-vitro evaluation of floating microballoons of indomethacin

    Acta Pol. Pharm.

    (2010)
  • R. Bodmeier et al.

    Spontaneous formation of drug-containing acrylic nanoparticles

    J. Microencapsul.

    (1991)
  • A. Brazma et al.

    Minimum information about a microarray experiment (MIAME)-toward standards for microarray data

    Nat. Genet.

    (2001)
  • H. Chen et al.

    Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development

    J. Cell Biol.

    (2003)
  • H.W. Chen et al.

    Titanium dioxide nanoparticles induce emphysema-like lung injury in mice

    FASEB J.

    (2006)
  • E. Corcelle et al.

    Disruption of autophagy at the maturation step by the carcinogen lindane is associated with the sustained mitogen-activated protein kinase/extracellular signal-regulated kinase activity

    Cancer Res.

    (2006)
  • H. Dika Nguea et al.

    Gene expression profile in monocyte during in vitro mineral fiber degradation

    Arch. Toxicol.

    (2008)
  • H. Eidi et al.

    Cytotoxicity assessment of heparin nanoparticles in NR8383 macrophages

    Int. J. Pharm.

    (2010)
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