Pharmaceutical nanotechnologyDrug delivery by polymeric nanoparticles induces autophagy in macrophages
Graphical abstract
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.
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