Hazard Assessment of Engineered Nanocarriers using in vitro and in vivo Studies

SUMMARY The lack of an accurate toxicity assessment for nanocarriers has led us to develop a multi-parametric assay that utilizes high-throughput screening (HTS) to provide rapid and cost effective ranking of materials. Currently, there is a poor understanding between the relationship of toxicity in vitro and in vivo. To this end, we have shown results of cell based cytotoxicity assays are comparable to the toxicity affects of nanocarriers during the development of Xenopus laevis (X. laevis). INTRODUCTION Nanotechnology is a rapidly evolving discipline within biomedicine. In particular, the development of nanoparticles (NPs) has had increasing interest due to their ability to combine targeted therapy with diagnostics. An accurate toxicity assessment is critical in order to progress the development of novel nanotherapies for clinical applications. Despite this, there is a lack of toxicity assessments available for the ever-increasing range of nanomaterials being developed. Cell based assays are often used to provide rapid and cost effective evaluation of nanoparticle toxicity. Although this can highlight any initial toxic properties of a novel particle, it fails to elucidate the biological response of the material in an intact system. A major hurdle in the development of a successful toxicity assay is the coupling of HTS cell based assays with the detail provided by assessment in a whole organism. Several cell based biochemical assays are being used to assess the cytotoxicity of a range of NPs. The results are comparable to toxicity assessments in vivo. We are using the African Clawed frog, X. laevis, as a model organism for the in vivo assessment of nanomaterials. Fish and amphibian models are fast and efficient whole systems that can be used in HTS. Furthermore, X. laevis has more phylogenetic similarities to humans than alternative models such as zebrafish, and are often used as a biassay tool for screening new compounds (1). By ranking NP toxicity from in vivo and in vitro results, we hope to develop a HTS assay that can be used to quickly assess the potential toxicity of any novel nanoparticle, before further screening using an expensive higher organism such as a mammalian model. We then aim to use this HTS for the assessment of NPs developed by us for targeted skin cancer therapy. EXPERIMENTAL METHODS Different NPs were selected for cell based cytotoxicity assessment: superparamegnetic iron oxide NPs (SPIONs); Cd-Se quantum dots (QDs); polystyrene NPs (PS); fluorescent polystyrene NPs (FS); silica NPs with COO and NH2 functional groups; and magnetic NPs (MNPs). All NPs were characterized by dynamic light scattering and transmission electron microscopy (TEM). For all cell based biochemical assays, the cell lines used were human skin melanoma (SKMel-28), human alveolar adenocarcinoma (A549) and Madin-Darby Canine Kidney (MDCK). Cell viability, after exposure to varying concentrations of NPs, was assessed using methyl thiazolyl tetrazolium bromide (MTT) assay. Further cytotoxicity assays will include the assessment of increased reactive oxygen species produced in response to NP stimulation, and initiation of apoptosis after NP stimulation. X. laevis were exposed to varying NP concentrations to assess the toxicity during different stages of development. Any resulting toxicity was recorded by scoring phenotypic abnormalities. Assessment of exposure, by cryosectioning of the X. laevis stimulated by fluorescent NPs, were used as evidence of NP uptake by the embryos. Further studies by inductively coupled plasma mass spectrometry (ICP-MS) and TEM will confirm uptake of the NPs. RESULTS AND DISCUSSION SK-Mel-28, A549 and MDCK cell lines all showed dose dependent loss of cell viability after 72 hours of stimulation with QDs and FS. MNP showed limited loss of cell viability in all cell lines. SPIONs have promising potential for both drug delivery and medical imaging and so their lack of toxicity in vitro is encouraging (figure 1). Figure 1. MTT assay showing SK-MEL-28, A549 and MDCK cell viability after stimulation of varying concentrations of FS, MNP, SPION and QD. Moreover, the NPs showed comparable toxicity levels in vivo. As expected, QDs caused phenotypic abnormalities including bent tail, bent spine and gut abnormalities. These results are consistent with toxicity affects of QDs in zebrafish (2). SPIONs showed no significant development abnormalities in X. laevis (figure 2). Figure 2. Phenotypes of embryos after treatment with increasing concentrations of QDs. (BS-bent spine, BTbent tail). 100% mortality was observed in embryos at late tail bud stage at concentrations as low as 1.5*10 NP/ml. CONCLUSION The combination of in vitro and in vivo toxicity assessment provides a powerful HTS assay that can be used to cheaply and efficiently gauge the potential toxicity of a novel NP. Hazard rankings of the NPs will be further validated in murine toxicity models.