The incidence of ocular pathologies is constantly increasing, as is the interest of the researchers in developing new strategies to ameliorate the treatment of these conditions. Nowadays, drug delivery systems are considered among the most relevant approaches due to their applicability in the treatment of a great variety of inner and outer eye pathologies through painless topical administrations. The design of such nanocarriers requires a deep study of many aspects related to the administration route but also a consideration of the authorities and pharmacopeial requirements, in order to achieve a clinical outcome. On such bases, the scope of this review is to describe the path of the analyses that could be performed on nanoparticles, along with the assessment of their applicability for ophthalmic treatments. Preformulation studies, physicochemical and technological characterization, and preliminary noncellular in vitro studies have been described in part 1 of this review. Herein, first the in vitro cellular assays are described; subsequently, nonocular organotypic tests and ex vivo studies are reported, as to present the various analyses to which the formulations can be subjected before in vivo studies, described in the last part. In each step, the models that could be used are presented and compared, highlighting the pros and cons. Moreover, their reliability and eventual acceptance by regulatory agencies are discussed. Hence, this review provides an overview of the most relevant assays applicable for nanocarriers intended for ophthalmic administration to guide researchers in the experimental decision process.
