Development of Lipid Coated Lanthanide Nanoparticle Reagents for High Sensitivity Mass Cytometry

L. Tong, J. Pichaandi, A. Bouzekri, O. Ornatsky, V. Baranov, M.A. Winnik
University of Toronto,
Canada

Keywords: lanthanide, nanoparticles, lipid encapsulation, surface modification, mass cytometry

Summary:

Low copy number proteins (fewer than 1000 copies per cell) play key roles in cellular functions in gene expression and signaling pathways. However, detection of these proteins remains to be one of the most challenging tasks in molecular biology. Recently, mass cytometry (MC), developed by our industrial partner Fluidigm Canada, is a cell-by-cell technique that uses metal tags for the detection of biomarkers. With metal tags, the sensitivity depends on the number of metal ions per antibody conjugate. Operationally, MC uses antibodies labeled with metal isotopes to detect proteins and other biomolecules on the cells. It measures antibody binding to the cells by atomic mass spectrometry, which detects metal isotopes and measures how many of each type are bound to each cell. Current reagents for biomarker detection by mass cytometry are metal-chelating polymers (MCPs) that are covalently attached to antibodies (Abs). Each Ab-MCP conjugate can be labeled with multiple copies of a different metal isotope (typically a lanthanide ion). However the number of lanthanide ions per MCP conjugate is 200 which can detect proteins with more than 10,000 copies. By incorporating more Ln ions per conjugate, one would be able to increase the sensitivity by several orders of magnitude. As an example, lanthanide nanoparticles (NPs) such as NaTbF4 NPs with a 10 nm diameter carry 8000 ions. To address the issue of sensitivity, we have developed a series of monodispersed lanthanide NPs (NaLnF4 where Ln: Sm to Ho, Y) between 10 to 30 nm in diameter. Since the as-synthesized Ln NPs are coated with hydrophobic ligands, one would need to transfer these NPs into aqueous environments. In our design, the new ligands for surface modification should provide colloidal stability in buffers, minimize interaction with cells and bear functional groups for bioconjugation. To satisfy these requirements, we employ lipid mixtures to encapsulate the Ln NPs via a monolayer and bilayer coating. The colloidal stability of the lipid encapsulated NPs was characterized by dynamic light scattering and electron microscopy. As well, the NPs were incubated with KG1a and Ramos cells and tested for non-specific binding by mass cytometry. Both the monolayer and bilayer coating show low levels of non-specific binding but the bilayer coating shows much better colloidal stability in saline buffers. These studies provide a basis for bioconjugation and testing of specific-binding to cells in the near future.