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Assay Cascade Protocols

The Nanotechnology Characterization Laboratory has developed a standardized analytical cascade that performs physicochemical characterization as well as preclinical testing of the immunology, pharmacology and toxicology properties of nanoparticles and devices. The NCL characterizes nanomaterials from academia, government, and industry, and will help transition them from the discovery-phase into clinical trials. NCL will work with the sponsor to help meet regulatory requirements for an Investigational New Drug (IND) or Investigational Device Exemption (IDE) filing with the FDA. Nanomaterial characterization, including physicochemical, in vitro, and in vivo characterization experiments, is conducted free-of-charge for accepted Assay Cascade proposals. The data generated from NCL characterization of a sponsor's nanomaterial can be used in regulatory filings, in publications, and to garner interest from investors.

The following are assays that have been standardized to work with a variety of nanomaterials. Many assays, however, must be individually tailored for each nanoparticle formulation (e.g. physicochemical analysis and animal studies). Therefore, this does not represent an exhaustive list of NCL capabilities. For more information on NCL assays and capabilities, please contact the NCL at

Sterility & Endotoxin

Microbial and endotoxin contamination can be common in a laboratory setting, and in many cases, contamination won’t significantly affect results of early development studies. However, many of the assays conducted at the NCL, especially immunological assays, can be. Immunological assays, especially, can be susceptible to microbial and/or endotoxin contamination, leading to misinterpretation of the data. Therefore, all materials submitted to the NCL undergo screening for these contaminants prior to any subsequent studies.

For additional information on endotoxin testing, please view the Journal of Visualized Experiments protocol here. You can also find several manuscripts on sterility and endotoxin testing on our bibliography page.

Detection of Endotoxin Contamination (Questions?)
STE-1.1 End point chromogenic LAL assay
STE-1.2 Kinetic turbidity LAL assay
STE-1.3 Gel-clot LAL assay
STE-1.4 Kinetic chromogenic LAL assay
Detection of Microbial Contamination (Questions?)
STE-2.1 Detection of microbial contamination using Millipore Sampler Devices
STE-2.2 Detection of bacterial contamination using LB agar plates
STE-2.3 Detection of bacterial contamination using tryptic soy agar plates
STE-2.4 Detection of bacterial contamination using tryptic soy agar plates & RPMI suspension for determination of bacterial ID
STE-3 Detection of mycoplasma contamination
Detection of β-Glucan Contamination (Questions?)
STE-4 Detection of β-glucan contamination

Physicochemical Characterization

Physical attributes such as size and ligand distribution, surface characteristics, composition, purity, and stability are key factors contributing to a nanomaterial’s in vivo behavior and tolerability. The second phase of the assay cascade therefore focuses on characterizing the material’s physical properties, including the particle’s size, size distribution, molecular weight, density, surface area, surface charge density, purity, surface chemistry, and stability. The batch-to-batch reproducibility of material as provided by the sponsor/vendor is also addressed during this stage. Many NCL physicochemical characterization assays are not listed here; most are individually tailored for each nanoparticle.

Liposomes, colloidial metal nanoparticles, and polymeric/polymeric prodrug nanoparticles are among the most common nanotechnology platforms used in drug delivery. To assist developers working with these platforms, the NCL has created an overview of the most critical characterization parameters as well as the techniques most commonly employed. Each nanoformulation is unique; as such, these are not intended to be a universal, standardized approach to characterization. Rather, they are intended to serve as an overview of a minimum set of parameters researchers should consider when developing their characterization portfolio.

Size, Size Distribution (Questions?)
PCC-1 Batch-Mode DLS
PCC-6 Atomic force microscopy
PCC-7 Transmission electron microscopy
PCC-10 Differential mobility analysis
PCC-15 High-resolution scanning electron microscopy
PCC-20 Particle Concentration & Size using the Spectradyne nCS1
PCC-21 Particle Size & Concentration of Metallic Nanoparticles using SP-ICP-MS
Nanoparticle Concentration (Questions?)
PCC-20 Particle Concentration & Size using the Spectradyne nCS1
PCC-21 Particle Size & Concentration of Metallic Nanoparticles using SP-ICP-MS
Surface Chemistry (Questions?)
PCC-2 Zeta Potential
PCC-16 Quantitation of PEG on PEGylated Gold Nanoparticles Using Reversed Phase High Performance Liquid Chromatography and Charged Aerosol Detection
PCC-17 Quantitation of Surface Coating on Metallic Nanoparticles Using Thermogravimetric Analysis
Chemical Composition (Questions?)
PCC-8 ICP-MS of Au in rat tissue (Supplement to PCC-8 & PCC-9)
PCC-9 ICP-MS of Au in rat blood (Supplement to PCC-8 & PCC-9)
PCC-11 Mass fraction of Au using ICP-OES
PCC-14 Free vs. chelated Gd using RP-HPLC‒ICP-MS
PCC-18 Quantitation of APIs in Polymeric Prodrug Formulations
Particle Fractionation (Questions?)
PCC-19 Asymmetric-Flow Field-Flow Fractionation
Solution Properties (Questions?)
PCC-12 Electrolytic conductivity
PCC-13 pH

In Vitro Characterization

Traditional in vitro biochemical analyses and cell-based assays can mimic the in vivo physiologic environment, addressing biological activity and toxicity issues. The NCL in vitro assay cascade is designed for quick evaluation of nanoparticles prior to the more cost- and labor-intensive in vivo studies. After the initial characterization of nanomaterials to assess their purity and functionality, their safety and efficacy are tested in vitro. The NCL in vitro cascade assesses binding and internalization of nanomaterials, blood contact properties such as coagulation, plasma protein binding, hemolysis, platelet aggregation, etc.

Hematology (Questions?)
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 PAGE
ITA-5.1 Complement activation by western blot (qualitative)
ITA-5.2 Complement activation by EIA (quantitative)
ITA-12 Plasma coagulation times
Immunology (Questions?)
ITA-6.1 Leukocyte proliferation (immunostimulation and immunosuppression)
ITA-6.2 Leukocyte proliferation (immunostimulation)
ITA-6.3 Leukocyte proliferation (immunosuppression)
ITA-7 NO- production
ITA-8.1 Chemotaxis
ITA-8.2 Chemotaxis using label-free, real time technology
ITA-9.1 Phagocytosis
ITA-9.2 Phagocytosis
ITA-10 Preparation of human whole blood and PBMC for analysis of cytokines, chemokines and interferons
ITA-22 IL-8 detection by ELISA
ITA-23 IL-1β detection by ELISA
ITA-24 TNF-α detection by ELISA
ITA-25 IFN-γ detection by ELISA
ITA-27 Multiplex ELISA for detection of cytokines, chemokines and interferons
ITA-11 Cytotoxic activity of NK cells by label-free RT-CES
ITA-14 Maturation of monocyte-derived dendritic cells
ITA-17 Leukocyte procoagulant activity
ITA-18 Human leukocyte proliferation (immunosuppression)
Mechanistic Immunotoxicology (Questions?)
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
Cancer Cell Biology (general) (Questions?)
IEA-1 Invasion Assay
Cytotoxicity (general) (Questions?)
GTA-1 MTT and LDH release in LLC-PK1 cells
GTA-2 MTT and LDH release in Hep G2 cells
Cytotoxicity (apoptosis) (Questions?)
GTA-5 Caspase 3 activation in LLC-PK1 cells
GTA-6 Caspase 3 activation in Hep G2 cells
GTA-14 Caspase3/7 activation in Hep G2 cells
Oxidative Stress (Questions?)
GTA-3 Glutathione assay in Hep G2 cells
GTA-4 Lipid peroxidation assay in Hep G2 cells
GTA-7 ROS assay in primary hepatocytes
Autophagy (Questions?)
GTA-11 MAP LC3I to LC3II conversion by western blot
GTA-12 Autophagic dysfunction in LLC-PK1 cells
Drug Release (Questions?)
PHA-1 Radioactive blood partitioning
PHA-2 Ultrafiltration using a stable isotope tracer

In Vivo Characterization

Pharmacology and Toxicology

NCL performs non-GLP* animal studies in rodents to determine ADME (absorption, distribution, metabolism and excretion) and toxicity profiles. NCL toxicology studies provide identification of target organs of acute and repeat-dose toxicity and may aid in the selection of starting doses for GLP preclinical and Phase I human trials. NCL ADME and pharmacokinetic (PK) studies track the various components of a nanoparticle formulation in blood and tissues and aid determination of tissue and systemic exposure, the routes and rates of clearance, and systemic half-life. In vivo ADME-toxicity studies are tailored for each individual nanoparticle.

*According to recent ICH guidelines, a non-GLP Single-Dose Acute Toxicity Study may be utilized in an IND/IDE filing with the US FDA, in conjunction with a GLP Repeat-Dose Toxicity Study.


NCL efficacy studies are conducted in rodents utilizing a variety of tumor models to provide independent verification of collaborators’ proof-of-concept studies. Efficacy of nanomaterial formulations can be tested using transgenic, orthotopic, xenograft, or metatstatic models. NCL has many different cell lines available, and can usually obtain approval for other cell lines as required for a specific nanotechnology strategy. In collaboration with the Frederick National Lab’s Small Animal Imaging Program (SAIP), we also have the capability to assess imaging efficacy using bioluminescence and fluorescence imaging, CT, MRI, and ultrasound.


NCL also has several in vivo methods established to assess the immunotoxicity of nanoformulations in rodents. These include tests for adjuvanticity, T-cell dependent antibody responses (TDAR), and the local lymph node assay (LLNA) / local lymph node proliferation (LLNP) test. Pyrogenicity of nanoformulations can also be assessed using an in vivo rabbit pyrogen test (RPT).

The Frederick National Laboratory for Cancer Research is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals (Health Research Extension Act of 1985, Public Law 99-158, 1986). Animal care is provided in accordance with the procedures outlined in the Guide for Care and Use of Laboratory Animals (National Research Council, 1996; National Academy Press, Washington, D.C.). All animal protocols are approved by the FNLCR institutional Animal Care and Use Committee.