University of Wollongong
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Quantification of ligand density and stoichiometry on the surface of liposomes using single-molecule fluorescence imaging

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posted on 2024-11-16, 05:46 authored by Lisa Belfiore, Lisanne SpenkelinkLisanne Spenkelink, Marie RansonMarie Ranson, Antonius van OijenAntonius van Oijen, Kara PerrowKara Perrow
Despite the longstanding existence of liposome technology in drug delivery applications, there have been no ligand-directed liposome formulations approved for clinical use to date. This lack of translation is due to several factors, one of which is the absence of molecular tools for the robust quantification of ligand density on the surface of liposomes. We report here for the first time the quantification of proteins attached to the surface of small unilamellar liposomes using single-molecule fluorescence imaging. Liposomes were surface-functionalized with fluorescently labeled human proteins previously validated to target the cancer cell surface biomarkers plasminogen activator inhibitor-2 (PAI-2) and trastuzumab (TZ, Herceptin®). These protein-conjugated liposomes were visualized using a custom-built wide-field fluorescence microscope with single-molecule sensitivity. By counting the photobleaching steps of the fluorescently labeled proteins, we calculated the number of attached proteins per liposome, which was 11 ± 4 proteins for single-ligand liposomes. Imaging of dual-ligand liposomes revealed stoichiometries of the two attached proteins in accordance with the molar ratios of protein added during preparation. Preparation of PAI-2/TZ dual-ligand liposomes via two different methods revealed that the post-insertion method generated liposomes with a more equal representation of the two differently sized proteins, demonstrating the ability of this preparation method to enable better control of liposome protein densities. We conclude that the single-molecule imaging method presented here is an accurate and reliable quantification tool for determining ligand density and stoichiometry on the surface of liposomes. This method has the potential to allow for comprehensive characterization of novel ligand-directed liposomes that should facilitate the translation of these nanotherapies through to the clinic.

Funding

RAPID: Collaborative Research: Multiscale plume modeling of the Deepwater Horizon oil-well blowout for environmental impact assessment and mitigation

Directorate for Engineering

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Functional Dissection of the Bacterial Replisome

Australian Research Council

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Citation

Belfiore, L., Spenkelink, L. M., Ranson, M., van Oijen, A. M. & Vine, K. L. (2018). Quantification of ligand density and stoichiometry on the surface of liposomes using single-molecule fluorescence imaging. Journal of Controlled Release, 278 80-86.

Journal title

Journal of Controlled Release

Volume

278

Pagination

80-86

Language

English

RIS ID

125925

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