Assay Cascade Protocols
National Cancer Institute
Nanotechnology Characterization Laboratory

Physico-Chemical Characterization					Method ID
	Size, Size Distribution
		Measuring the Size of Nanoparticles in Aqueous Media Using Batch-Mode DLS			NIST-NCL PCC-1
	Topology Molecular Weight Aggregation Purity Chemical Composition Surface Characteristics Functionality Zeta Potential Stability Solubility
In Vitro					Method ID
	Sterility
		Endotoxin			STE-1
		Bacterial/Yeast/Mold			STE-2
		Mycoplasma			STE-3
	Targeting
		Cell Binding/Internalization
	In Vitro Immunology
		Blood Contact Properties
			Plasma Protein Binding (2D PAGE)		ITA-4
			Hemolysis		ITA-1
			Platelet Aggregation		ITA-2
			Coagulation		ITA-12
			Complement Activation		ITA-5
		Cell-Based Assays
			CFU-GM		ITA-3
			Leukocyte Proliferation		ITA-6
			Macrophage/Neutrophil Function (4 categories):
				Phagocytosis	ITA-9
				Cytokine Induction (6 assays): TNFα, IL-1β, IL-6, IL-8, IL-10, IL-12	ITA-10
				Chemotaxis	ITA-8
				Oxidative Burst (NO)	ITA-7
			Cytotoxic Activity of NK Cells		ITA-11
	Toxicity
		Phase I/II Enzyme Induction/Supression
		Oxidative Stress
			GSH Homeostasis (HEP G2)		GTA-3
			Lipid Peroxidation (HEP G2)		GTA-4
			ROS		GTA-7
		Cytotoxicity (necrosis)
			MTT and LDH Release (porcine renal proximal tubule cell)		GTA-1
			MTT and LDH Release (HEP G2)		GTA-2
		Cytotoxicity (apoptosis)
			Caspase 3 Activation (porcine renal proximal tubule cell)		GTA-5
			Caspase 3 Activation (HEP G2)		GTA-6
	Metabolic Stability
		CYP450 (Phase 1)
		Glucuronidation, Sulphation (Phase II)
In Vivo
	Initial disposition study
		Tissue Distribution
		Clearance
		Half-life
		Systemic exposure (plasma AUC)
	Immunotoxicity
		28 day screen
		Immunogenicity (repeat-dose tox study)
	Dose -Range Finding Toxicity
		Blood Chemistry
		Hematology
		Histopathology
		Gross Pathology
	Efficacy
		Therapeutic
		Imaging
	GLP Studies
		PK/ADME
		Expanded Single Dose Acute Tox