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Protocols and Capabilities from the Nanotechnology Characterization Lab

The Nanotechnology Characterization Laboratory (NCL) 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. These efforts are meant to aid developers in transitioning their concepts from the discovery-phase into clinical trials. NCL will work with each sponsor to individually tailor a research plan specific to their concept, to help fill critical gaps in their characterization portfolio as they prepare to meet regulatory requirements for an Investigational New Drug (IND) or Investigational Device Exemption (IDE) filing with the FDA. In this video, Dr. Stephan Stern talks about the importance of using standardized methods for nanomedicine testing in pursuit of regulatory filings.  

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, or for questions on any of the protocols, please contact the NCL at

In addition to protocols found here, the Frederick National Lab's STAR TREC initiative has created a hub to share standards and references in a variety of disciplines, including nanotechnology research, in an effort to promote scientific reproducibility of data. 

Agar plate, nanoparticle, blood vessel and mouse with tumor used as illustrative depiction of the areas of NCL characterization: sterility, physicochemical characterization, immunology and pharmacology/toxicology.

The NCL's Assay Cascade provides for characterization in four primary areas: sterility, endotoxin & beta-glucans; physicochemical characterization; in vitro and in vivo immunology; and in vitro and in vivo pharmacology and toxicology.

Sterility & Endotoxin Protocols

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 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.

To aid developers struggling with these contaminants, the NCL has prepared a guide with tips on how to test for sterility and endotoxin levels, minimize contamination during preparation, and remove/reduce endotoxin levels in samples. In addition, the NCL has published a video in the Journal of Visualized Experiments with recommendations on performing the various Limulus Amoebocyte Lysate (LAL) assays for detection of endotoxin.

Detection of Endotoxin Contamination

Detection of Microbial Contamination

  • 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

  • STE-4  Detection of β-glucan contamination

Physicochemical Characterization Protocols

Physical attributes are key factors contributing to a nanomaterial’s in vivo behavior and tolerability. Using state-of-the-art instrumentation, nanoparticles are subjected to a thorough assessment of their physical and chemical properties, including the particle’s size, size distribution, molecular weight, morphology, surface characteristics, composition, and purity. In addition to evaluating direct traits of the particle, aspects such as stability, lot-to-lot reproducibility, as well as assessment of the starting materials are also critical components of the characterization process.

Many physicochemical characterization assays are individually tailored for each nanoparticle; therefore, this does not represent an exhaustive list of assays available for the physicochemical characterization of the various nanoparticle platforms. NCL will work alongside investigators to understand the intricacies of their formulation and develop a testing plan that fills any critical gaps in their knowledge and understanding of the formulation.

Among the most common nanotechnology platforms used in drug delivery are liposomes, colloidal metal nanoparticles, and polymeric/polymeric prodrug  nanoparticles. 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. 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

Dynamic Light Scattering - Nanotechnology Characterization Lab

A brief introduction to measuring nanoparticle size/size distribution using batch-mode dynamic light scattering.

  • 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 size & concentration using the Spectradyne nCS1
  • PCC-21  Particle size & concentration of metallic nanoparticles using SP-ICP-MS

Solution Properties

Surface Chemistry

  • PCC-2  Zeta potential
  • PCC-16  Quantitation of PEG on PEGylated gold nanoparticles using RP-HPLC and charged aerosol detection
  • PCC-17  Quantitation of surface coating on metallic nanoparticles using thermogravimetric analysis

Chemical Composition

Nanoparticle Concentration

  • PCC-20  Particle size & concentration using the Spectradyne nCS1
  • PCC-21  Particle size & concentration of metallic nanoparticles using SP-ICP-MS

Particle Fractionation

  • PCC-19  Asymmetric-flow field-flow fractionation

Immunology Protocols

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 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-PAGE
  • ITA-5.1  Complement activation by western blot (qualitative)
  • ITA-5.2  Complement activation by EIA (quantitative)
  • ITA-12  Plasma coagulation times

In Vitro Immunology

  • ITA-3  CFU-GM
  • 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  Nanoparticle effects on monocyte/macrophage phagocytic function
  • 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  TNFα detection by ELISA
  • ITA-25  IFNγ 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 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 reported cell lines

In Vitro Cancer Cell Biology

In Vivo Immunotoxicity 

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).

Pharmacology & Toxicology Protocols

A thorough understanding of nanomedicine pharmacology and toxicology is essential to identify liabilities and optimize drug formulation. Pharmacology and toxicology properties are frequently not identified until in vivo preclinical studies are performed later in the development process. There are, however, several predictive in vitro assays that can be extremely informative for development of nanomedicines, such as drug release in physiologically relevant matrices, cytotoxicity in cell lines relevant to a specific indication, and assays to evaluate mechanisms of toxicity.

In addition to these in vitro studies, the NCL also provides in vivo pharmacokinetic and toxicity studies of nanomaterials. In vivo studies are conducted in collaboration with the Frederick National Laboratory’s Laboratory Animal Sciences Program, which includes the Molecular Histopathology Laboratory and Small Animal Imaging Program, among others. All in vivo studies are individually tailored for each tested nanoparticle and are collaboratively agreed upon with the developer before testing begins; in vivo studies are designed to mimic the intended clinical dose (adjusted for species), dosing regimen, and route of administration.

In Vitro Cytotoxicity (general)

  • GTA-1  MTT & LDH release in LCC-PK1 cells
  • GTA-2  MTT & LDH release in Hep G2 cells

In Vitro Cytotoxicity (apoptosis)

  • GTA-5  Caspase 3 activation in LLC-PK1 cells
  • GTA-6  Caspase 3 activation in Hep G2  cells
  • GTA-14  Caspase 3/7 activation in Hep G2 cells

In Vitro Oxidative Stress

  • GTA-3  Glutathione assay in Hep G2 cells
  • GTA-4  Lipid peroxidation assay in Hep G2 cells
  • GTA-7  ROS assay in primary hepatocytes

In Vitro Autophagy

  • GTA-11  MAP LC3I to CL3II conversion by western blot
  • GTA-12  Autophagic dysfunction in LLC-PK1 cells

In Vitro Drug Release

  • PHA-1  Radioactive blood partitioning
  • PHA-2  Ultrafiltration using a stable isotope tracer

In Vivo 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 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 systemic and tissue 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.

In Vivo Efficacy

NCL efficacy studies are conducted in rodents utilizing a variety of tumor models to provide independent verification of collaborators’ proof-of-concept studies, or add additional pharmacology endpoints to the original study. Efficacy of nanomaterial formulations can be tested using transgenic, syngeneic, xenograft, orthotopic or metastatic 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, PET, MRI, and ultrasound.


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. 

Capabilities & Instrumentation

Below is a representative list of the capabilities and instruments commonly utilized by the NCL in the characterization of submitted nanomaterials; this is not an exhaustive list. 

The mention of trade names and manufacturers is for informational purposes only. The NCL does not endorse any of the suppliers listed below. Equivalent instrumentation from alternate vendors can be substituted.

Chemical Characterization

  • Atomic force microscopy (AFM)
  • Reaction microwave
  • Chromatography (FPLC, HPLC, GC, and UHPLC) with various detection capabilities (diode array UV-vis, fluorescence, refractive index, charged aerosol, mass spectrometry)
  • Differential scanning calorimetry (DSC)
  • Dynamic light scattering (DLS)
  • Electrochemical workstation
  • Electron microscopy (transmission, cryo-transmission, scanning) with energy dispersive X-ray (EDS) detector
  • Elemental analysis (C, H, N, O, S)
  • Asymmetric-flow field-flow fractionation (AF4) with various detectors (diode array UV-vis, multi-angle light scattering, refractive index, viscometry and dynamic light scattering)
  • Mass spectrometry (single quad, triple quad, Orbitrap, inductively coupled plasma)
  • Laser diffraction
  • LV1 Microfluidizer
  • pH Titrator
  • Quartz crystal microbalance with dissipation (QCM-D)
  • Single particle counting and sizing based on resistive pulse sensing and light scattering
  • Spectroscopy (UV-vis, fluorescence, infrared, Raman)
  • Spin Coater
  • Thermogravimetric analysis (TGA)

Biological Characterization

  • Cell counters
  • Coagulometer
  • CHRONO-LOG Model 700 Whole Blood/Optical Lumi-Aggregometer
  • Flow cytometers
  • Fluorescent microscope
  • Freeze dryers
  • GentleMACS dissociator 
  • Imaging systems
  • Kinetic tube reader
  • Liquid scintillation counter
  • Neon transfection system
  • Optical microscopes
  • Plate readers
  • Real-time cell analyzers
  • Thermal cyclers

Frederick National Lab Instrumentation

In addition to collaborative resources located at NIST, the NCL also has direct access to all resources, instruments, and expertise within the Frederick National Laboratory for Cancer Research. Some examples include a mass spectrometry facility, nuclear magnetic resonance spectrometers, an optical microscopy laboratory, additional electron microscopy capabilities, a proteomics laboratory, a sequencing facility, and an animal research facility complete with pathology, histology, and a full range of animal imaging capabilities.

For more information on the added resources available, please visit the Frederick National Lab website.

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