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National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]. Bethesda (MD): National Cancer Institute (US); 2005 May 1-. doi: 10.17917/2MYC-DE76

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National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet].

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Parameters, Methods and Considerations for the Physicochemical Characterization of Colloidal Metal Nanoparticles

Published: .

Size/Size Distribution

  • Dynamic light scattering (DLS)
  • Multi-angle light scattering (MALS)
  • Laser diffraction
  • Transmission electron microscopy (TEM)
  • Resistive pulse sensing
  • Asymmetric-flow field-flow fractionation (AF4)-MALS/DLS
  • Single particle inductively-coupled plasma mass spectrometry (spICP-MS)
Three electron micrographs showing metallic nanoparticles existing in monomer, dimer and trimer aggregation states. Single particle ICP-MS traces are also shown for the three samples.

Single particle ICP-MS and representative TEM images of PEGylated core-shell nanoparticles. spICP-MS can resolve the different species in a single run and determine the size of each population.

Composition

  • Metal concentration: total & free
  • Coating (polymer, protein, lipid) concentration: total & free
  • Targeting ligand concentration: total & free
  • Excipient concentrations
  • Particles per mL concentration
  • Osmolality, viscosity measurements
Schematic showing the decomposition of a gold nanoparticle into its components, protein, lipids and gold cyanide. Fluoresnce trace is shown, used for detection and quantitation of protein. HPLC trace is shown, used for detection and quantitation of the lipids.

Colloidal gold nanoparticles are dissolved using KCN to afford free protein and lipid surface ligands. The resulting solution is analyzed for concentration of individual components.

Surface Characteristics

  • Zeta potential
  • Protein binding assessment by AF4-MALS/DLS
  • Quartz crystal microbalance with dissipation monitoring (QCM-D)
Schematic showing gold nanoparticle being conjugated with PEG and protein. Zeta potential traces for each sample are also shown.

Monitoring the reaction progress of PEG and protein grafting to the surface of colloidal gold nanoparticles by zeta potential. Measurement conditions were identical for all samples.

Purity

  • Metal impurities
  • Shape distribution (e.g., spheres versus rods)
  • Coating impurities
  • Residual solvents and reagents
Flow-mode DLS trace showing fractionation of the peak. electron micrographs are also shown for each fraction, depicting a change in the shape of particles collected in each fraction.

Purity assessment of PEGylated gold nanorods as defined by the presence of non-rod shaped particles. Particles were separated by AF4-DLS, and collected fractions were analyzed by TEM to confirm shape. Adapted from Anal Bioanal Chem, 2020, 412(2), 425-428.

Batch-to-Batch Consistency

  • Assessed by choosing relevant parameters (i.e., lot release criteria) that relate to a desired in vivo outcome
Electron micrographs of three lots of the same metallic nanoparticle. AF4-MALS spectra for each is also shown. No difference is noted in the electron micrographs, yet a clear difference can be seen in the MALS spectra.

Representative TEM images (top) versus flow-mode AF4-MALS (bottom) measurements for three batches of a polymer coated metal oxide formulation. In this case, AF4-MALS was better suited in assessing the size distribution and differences between these three lots. Adapted from J Control Release, 2019, 299, 31-43.

Stability

  • Size/Size distribution; aggregation
  • Free metal ion release
  • Coating release
  • Stability in plasma
  • Solvent, thermal, pH, photo, freeze-thaw, lyophilization, centrifugation, filtration
  • Storage conditions/shelf-Life
Schematic of the stirred cell apparatus. Accompanying graph shows the accumulation of free silver over time, as a measure of shelf life for silver nanoparticles.

Stability assessment of four batches of colloidal silver nanoparticles as determined by free silver concentrations. Stirred cell filtration was used to separate free silver from nanoparticle silver. The permeate silver concentration was measured by ICP-MS.

Starting Material Characterization

  • Colloidal metal NPs: size, shape, composition & purity
  • Coating : molecular mass, polydispersity Index, purity, functionality
  • Storage conditions/shelf-life
HPLC traces for two samples of PEG, showing a different degree of impurities in each.

Purity and functionality characterization of 20 kDa mPEG-SH from two manufacturers. Purity was assessed by RP-HPLC-CAD and thiol content by the Ellman’s reagent. Adapted from AAPS J, 2017, 19(1), 92-102.

Morphology

  • Transmission electron microscopy (TEM)
Electron micrographs of gold-core silica-shell nanoparticles.

Representative TEM images of gold-core silica-shell nanoparticles. In addition to determining the size distribution, TEM can be used to evaluate morphology and purity assessment. Here, smaller gold nanoparticles and incomplete gold coating were observed. Elemental composition was confirmed by energy dispersive spectroscopy (EDS).

Relevant NCL Publications

  1. Anal Bioanal Chem, 2020, 412(2), 425–428. PMID: 31776639 [PubMed: 31776639]
  2. J Control Release, 2019, 299, 31–43. PMID: 30797868 [PubMed: 30797868]
  3. Pharmaceutical Research, 2019, 37, 6. PMID: 31828540 [PMC free article: PMC7274461] [PubMed: 31828540]
  4. Methods in Molecular Biology, Vol. 1628, 2018, p. 37–47. PMID: 29039091 [PubMed: 29039091]
  5. Methods in Molecular Biology, Vol. 1628, 2018, p. 49–55. PMID: 29039092 [PubMed: 29039092]
  6. Methods in Molecular Biology, Vol. 1628, 2018, p. 57–63. PMID: 29039093 [PubMed: 29039093]
  7. Anal Bioanal Chem, 2017, 409(24), 5779–5787. PMID: 28762066 [PubMed: 28762066]

About NCL

The Nanotechnology Characterization Laboratory (NCL) is a resource for nanotech researchers and organizations developing nano-based therapies and diagnostics. The NCL provides preclinical characterization services through various collaboration mechanisms. Learn more by visiting our website: https://ncl​.cancer.gov

Email us at vog.hin.liam@lcn

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