Multicolor flow cytometry-based immunophenotyping for preclinical characterization of nanotechnology-based formulations: an insight into structure activity relationship and nanoparticle biocompatibility profiles.

Introduction: Immunophenotyping, which is the identification of immune cell subsets based on antigen expression, is an integral technique used to determine changes of cell composition and activation in various disease states or as a response to different stimuli. As nanoparticles are increasingly utilized for diagnostic and therapeutic applications, it is important to develop methodology that allows for the evaluation of their immunological impact. Therefore, the development of techniques such as immunophenotyping are desirable. Currently, the most common technique used to perform immunophenotyping is multicolor flow cytometry. Methods: We developed two distinct multicolor flow cytometry immunophenotyping panels which allow for the evaluation of the effects of nanoparticles on the composition and activation status of treated human peripheral blood mononuclear cells. These two panels assess the presence of various lymphoid and myeloid-derived cell populations as well as aspects of their activation statuses-including proliferation, adhesion, co-stimulation/presentation, and early activation-after treatment with controls or nanoparticles. To conduct assay performance qualification and determine the applicability of this method to preclinical characterization of nanoparticles, we used clinical-grade nanoformulations (AmBisome, Doxil and Feraheme) and research-grade PAMAM dendrimers of different sizes (G3, G4 and G5) and surface functionalities (amine-, carboxy- and hydroxy-). Results and Discussion: We found that formulations possessing intrinsic fluorescent properties (e.g., Doxil and AmBisome) interfere with accurate immunophenotyping; such interference may be partially overcome by dilution. In the absence of interference (e.g., in the case of dendrimers), nanoparticle size and surface functionalities determine their effects on the cells with large amine-terminated dendrimers being the most reactive.