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

Hannah S Newton; Jenny Zhang; Duncan Donohue; Ragi Unnithan; Edward Cedrone; Jie Xu; Alison Vermilya; Tyler Malys; Jeffrey D Clogston; Marina A Dobrovolskaia

doi:10.3389/falgy.2023.1126012

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

Front Allergy. 2023 Jul 4:4:1126012. doi: 10.3389/falgy.2023.1126012. eCollection 2023.

Affiliations

¹ Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research Sponsored by the National Cancer Institute, Frederick, MD, United States.
² Agilent Technologies, Santa Clara, CA, United States.
³ Statistics Department, Frederick National Laboratory for Cancer Research Sponsored by the National Cancer Institute, Frederick, MD, United States.

Abstract

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.

Keywords: flow cytometry; immunophenotyping; lymphocytes; monocytes; nanomaterials; nanoparticles.

Grants and funding

This study was funded by federal funds from the National Cancer Institute, National Institutes of Health, under contract 75N91019D00024. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.