Evaluation of nanoformulated therapeutics in an ex-vivo bovine corneal irritation model Sriramoju Bhasker, Roy Kislay, Kanwar K. Rupinder, Kanwar R. Jagat ⇑ Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), Molecular and Medical Research (MMR) Strategic Research Centre, School of Medicine (SoM), Faculty of Health, Deakin University, Waurn Ponds, Victoria 3217, Australia article info Article history: Received 17 September 2014 Accepted 18 January 2015 Available online 13 February 2015 Keywords: Corneal irritation PLGA Chitosan SurR9-C84A Bovine lactoferrin abstract Aim: To determine the internalization and protective effects of potential ophthalmic formulations and nanoformulated natural proteins in ex-vivo bovine corneal alkali burn model. Methods: The bovine cornea obtained were subjected to the 0.5 N NaOH insult that induced alkali burn and inflammation as observed in the in vivo situation. The toxic effects of the nanoformulation were evaluated in the normal and insult induced cornea using histological analysis. Internalization studies were carried out using in vivo imaging and analysis (IVIS, PerkinElmer, USA). Results: The nanoformulations employed in this study showed no obvious changes in the integrity of the cornea. Further, improvements in the light transmittance and reduced inflammation were observed. The IVIS showed a dose dependant increase in the uptake of the nanoformulations with time. Conclusion: The nanoformulated bovine lactoferrin and SurR9-C84A (SR9) proteins evaluated in the ex vivo bovine corneal irritation model is the first of its kind, and we report here the non-toxic and thera- peutic potential of these formulations for topical applications. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction In order to replace the in vivo eye test, several efforts have been made to find an apt replacement, and Draize et al., were the first to report several methods for studying irritation and toxic effects of substances applied topically (Draize et al., 1944). The same proce- dure was also made as a governmentally endorsed methodology and is described in the organisation for economic co-operation and development (OECD) testing guidelines 405 (OECD, 2002). The first in vitro alternative methodology to replace the in vivo eye test was suggested by Fraizer et al., 1987. In vitro methodolo- gies such as isolated rabbit eye (IRE) test proposed by Burton et al., 1981 and bovine corneal opacity (BCOP) assay proposed by Gautheron et al. (1992) have been used for the prediction of ocular irritation of the prototype shampoo formulations (Cooper et al., 2001). The BCOP test is performed to evaluate the response of an excised bovine cornea upon exposure to potential ocular irritants by measuring the measure light transmission and permeability of the corneal layer. Histological evaluations provide a better characterisation of substances that do not produce measurable opacity or permeability changes (Curren et al., 1999). Several other studies have also made use of these assays to determine the pharmacological screening of compounds such as benzimidazoles (with analgesic and anti-inflammatory properties) (Jesudason et al., 2009). A marked reduction in the opacity was reported after treatment with 1% NaOH for mere 30 s to 1 min. On the basis of A570 score, NaOH was classified as a mild irritant that can lead to an opacity score in the moderate to severe irritant range (Ubels et al., 2000). Another study reported evaluation of fifty-six chemicals including alcohols, surfactants and esters using the BCOP assay. An accuracy rate of 69.6% was obtained using the results with a favourable predictive capacity having the accuracy of 71.4% (Hayashi et al., 2012). Normally, the opacity is measured using a spectrometer or an OP-KIT opacitometer that provides a centre-weighted reading of light transmission through the corneal tissue. However, studies have been performed, showing that a laser-based opacitometer allows the analysis for complete corneal surface and thus, has an improved sensitivity to detect any minute changes in the corneal transparency (Van Goethem et al., 2010). The potential cytotoxicity of nanoparticles is a rising concern on account of their agglomera- tion, stability issues, dosage frequency, etc. Thus, studies have shown that BCOP and several other assays may be useful to deter- mine the irritation and cytotoxic effects of nanoparticles in vitro in non-cancerous cells (Hartung, 2010). Another study has used BCOP test to determine the toxic effects of zinc-oxide (Zn-O) http://dx.doi.org/10.1016/j.tiv.2015.01.007 0887-2333/Ó 2015 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +61 3 5227 1148; fax: +61 3 5227 3402. E-mail address: jagat.kanwar@deakin.edu.au (K.R. Jagat). Toxicology in Vitro 29 (2015) 917–925 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit