Assay Cascade Protocols

The Nanotechnology Characterization Laboratory has developed a standardized analytical cascade that tests the preclinical toxicology, pharmacology, and efficacy 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. NCL preclinical characterization, including physicochemical, in vitro, and in vivo characterization experiments, is available free-of-charge to accepted Assay Cascade proposals, and NCL data 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 ncl@mail.nih.gov.

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.

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.

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.

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 preliminary identification of target organs of acute toxicity and may aid in the selection of starting doses for Phase I human trials. NCL ADME and pharmacokinetic (PK) studies track the various components of a nanoparticle formulation in blood and tissues and may help determine the route of clearance and tissue residence times. 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 Acute Toxicity Study.

Efficacy

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.

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).

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.