In Vitro Target Organ Toxicity

A recently published report from the International Life Sciences Institute Research Foundation/Risk Science Institute Nanomaterial Toxicity Screening Working Group73 recommends the inclusion of several specific in vitro assays in a standard protocol of safety assessment. Much of the report focused on toxicity screening for environmental exposure to nanoparticles and thus emphasized environmentally relevant exposure routes. However, in addition to the in vitro examination of so-called portal-of-entry tissues, the report expressed the need for inclusion of potential target organs. The liver and kidney were selected as ideal candidates for these initial in vitro target organ toxicity studies, since preliminary investigations (discussed below) have identified these as the primary organs involved in the accumulation, processing, and eventual clearance of nanoparticles.

The liver has been identified in many studies as the primary organ responsible for reticuloendothelial capture of nanoparticles, often due to phagocytosis by Kupffer cells.65-67 Fluor-escein isothiocyanate-labeled polystyrene nanoparticles and radiolabeled dendrimers, for example, are rapidly cleared from the systemic circulation by hepatic uptake following intravenous injection.4,68 Hepatic uptake has also been shown to be a primary mechanism of hepatic clearance for parenterally administered fullerenes, dendrimers, and quantum dots.20,69,70 In addition to hepatic accumulation, nanoparticles have also been shown to have a detrimental effect on liver function ex vivo and alter hepatic morphology. Hepatocytes isolated from rats intravenously administered polyalkylcyanoacrylate nanoparticles had diminished secretion of albumin and decreased glucose production.71 This alteration in albumin synthesis was also observed in freshly isolated rat hepatocytes exposed to polyalkylcyanoacrylate nanoparticles. Dendrimers have also been shown to cause liver injury. Repeated dosing of mice with polyamidoamine (PAMAM) dendrimers resulted in vacuolization of the hepatic parenchyma, suggesting lysosomal dysfunction.72

Sprague-Dawley rat hepatic primary cells and human hepatoma Hep-G2, selected for in vitro hepatic target organ toxicity assays, have a long history of use in toxicological evaluation.73-75 Hep-G2 cells were chosen since they are a readily available hepatocyte cell line with high metabolic activity.76 Rat hepatic primary cells were also chosen for toxicological studies, since hepatic primary cells in culture are more reflective of in vivo hepatocytes with regard to enzyme expression and specialized functions. One survey found rat hepatic primary cells up to ten times more sensitive to model hepatotoxic agents than established hepatic cell lines.77 Rat hepatic primaries represent a suitable alternative to human hepatic primary cells, which are scarce, costly, and suffer from interindividual variability.

Preliminary pharmacokinetic studies of parenterally administered, radiolabeled carbon nano-tubes, dendritic fullerenes, and low generation dendrimers in rodents have identified urinary excretion as the principal mechanism of clearance.78-80 A variety of engineered nanoparticles, including actinomycin D-loaded isobutylcyanoacrylate, doxorubicin-loaded cyanoacrylate, and dendrimer nanoparticles, have also been shown to distribute to renal tissue following parenteral

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administration in rodents. , , In the case of the doxorubicin-loaded cyanoacrylate nanoparticles, doxorubicin renal distribution was increased due to capture of the nanoparticles by glomerular mesangial cells. This resulted in a shift in the primary target organ from the heart to the kidney. Doxorubicin-induced renal injury presented as a severe proteinuria. Kidney injury has been demonstrated for other nanomaterials as well. Nano-zinc particles, for example, caused severe histological alterations in murine kidneys and Q-dots were shown to be cytotoxic to African green monkey kidney cells.83,84

The porcine renal proximal tubule cell line, LLC-PK1, was selected as a representative kidney cell line, since it is readily available through ATCC and has been used extensively in nephrotoxicity screening and mechanistic studies.85 The SD rat hepatic primary, LLC-PK1 and Hep-G2 cells are adherent, which can simplify sample preparation, and can be propagated in a 96-well plate format suitable for high-throughput screening. The 96-well format allows for detailed concentration-response curves and multiple controls to be run on the same microplate. These cell lines were subjected to a variety of in vitro assays, described below, to evaluate cytotoxicity, mechanistic toxicology, and pharmacology. These assays have been selected primarily for their superior performance, convenience, and adaptability in evaluating this new class of biomedical agent.

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