By Nanotechnology Characterization Laboratory (NCL)
Nanoformulated drugs often induce a variety of toxicities and reactions originating from nanoparticle interaction with various components of the immune system. Overt cytokine release, complement activation, leukocyte responses, and perturbation of blood coagulation pathways are among the most frequent reasons halting the preclinical development of nanomedicines. Therefore, screening for these toxicities early in the development process can not only save the developer resources but can also prevent adverse reactions in patients when the formulation reaches clinical trials. NCL's immunological characterization of nanomedicines aims to do just this. It provides analysis of not only the nanoformulation, but in certain cases, precursor formulations, control formulations, individual components, and formulation constituents. Characterization spans both in vitro and in vivo characterization and includes in vitro hematological compatibility, in vitro immunotoxicity, and evaluation of in vivo immunotoxicity. The assays selected for your formulation will be chosen based on current knowledge about the nanoplatform, the active pharmaceutical ingredient, and the critical gaps in your developmental path. In addition to the detailed protocols offered below, we have developed a guide that can be consulted for development of an immunogenicity assay. Since many aspects of these assays are specific to each project, a guide—rather than a standardized, step-by-step protocol—is offered to help understand the general principles, identify the most critical steps, and provide links to useful resources. In Vitro Hematology: ITA-1 Hemolysis; ITA-2.1 Platelet aggregation by cell counting; ITA-2.2 Platelet aggregation by light transmission; ITA-4 Interaction with plasma proteins by 2D-polyacrylamide gel electrophoresis; ITA-5.1 Complement activation by western blot (qualitative); ITA-5.2 Complement activation by enzyme immunoassay (quantitative); ITA-12 Plasma coagulation times. In Vitro Immunology: ITA-3 Granulocyte-macrophage colony-forming units; ITA-6.1 Leukocyte proliferation (immunostimulation and immunosuppression); ITA-6.2 Leukocyte proliferation (immunostimulation); ITA-6.3 Leukocyte proliferation (immunosuppression); ITA-7 Nitrite- production; ITA-8.1 Chemotaxis; ITA-8.2 Chemotaxis using label-free, real-time technology; ITA-9.1 Phagocytosis; ITA-9.2 Nanoparticle effects on monocyte/macrophage phagocytic function; ITA-10 Preparation of human whole blood and peripheral blood mononuclear cells for analysis of cytokines, chemokines, and interferons; ITA-22 IL-8 detection by enzyme-linked immunosorbent assay; ITA-23 IL-1β detection by enzyme-linked immunosorbent assay; ITA-24 TNFα detection by enzyme-linked immunosorbent assay; ITA-25 IFNγ detection by enzyme-linked immunosorbent assay; ITA-27 Multiplex enzyme-linked immunosorbent assay for detection of cytokines, chemokines and interferons; ITA-11 Cytotoxic activity of natural killer cells by label-free real-time cell electronic system; ITA-14 Maturation of monocyte-derived dendritic cells; ITA-17 Leukocyte procoagulant activity; ITA-18 Human lymphocyte activation (immunosuppression). In Vitro Mechanistic Immunotoxicology: ITA-26 Detection of intracellular complement activation in human T lymphocytes; ITA-31 Detection of nanoparticle-mediated total oxidative stress in T cells using CM-H2DC-FDA dye; ITA-32 Detection of mitochondrial oxidative stress in T cells using MitoSOX Red dye; ITA-33 Detection of changes in mitochondrial membrane potential in T cells using JC-1 dye; ITA-34 Detection of antigen presentation by murine bone marrow-derived dendritic cells; ITA-35 Antigen-specific stimulation of CD8+ T-cells by murine bone marrow-derived dendritic cells; ITA-36 Detection of naturally occurring antibodies to PEG and PEGylated liposomes; ITA-29 Detection of nanoparticles' ability to stimulate toll-like receptors using HEK-Blue reporter cell lines; ITA-37.1 Immunophenotyping: Instrument calibration and reagent qualification for immunophenotyping analysis of human peripheral blood mononuclear cells; ITA-37.2 Immunophenotyping: Analysis of nanoparticle effects on the composition and activation status of human peripheral blood mononuclear cells; ITA-38 IgE-Dependent Mast Cell Degranulation; ITA-40 Understanding the role of scavenger receptor A1 in nanoparticle uptake by murine macrophages; ITA-41 Detection of IFNγ using ELISpot; ITA-42 Detection of IFNγ and IL-2 using FLUOROSpot. In Vitro Cancer Cell Biology: IEA-1 Invasion assay. In Vivo Immunotoxicity: NCL also has several in vivo methods established to assess the immunotoxicity of nanoformulations in rodents. These include tests for adjuvanticity, immunosuppression (T-cell dependent antibody response test), delayed type hypersensitivity (the local lymph node assay/local lymph node proliferation test), myelosuppression (CFU-GM), autoimmunity (SJL/J lupus model), and autoinflammation (psoriasis model).
