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Mapping Antibody Domain Exposure on Nanoparticle Surfaces Using DNA-PAINT.

Marrit M E Tholen¹, Bas J H M Rosier¹, Robin T Vermathen¹

¹Department of Biomedical Engineering, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, 5612 AZ Eindhoven, The Netherlands.

|June 7, 2023

Summary

This summary is machine-generated.

This study introduces a new super-resolution microscopy method to precisely measure antibody orientation on nanoparticles. This technique quantifies both fragment antibody-binding (Fab) and fragment crystallizable (Fc) domain exposure, crucial for targeted nanomedicine.

Keywords:

DNA-PAINT antibodies heterogeneity nanomedicine nanoparticles super-resolution microscopy

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Area of Science:

Nanotechnology
Bioconjugation Chemistry
Super-resolution Microscopy

Background:

Antibody (Ab) decoration of nanoparticles is vital for targeted drug delivery and imaging.
Antibody orientation on nanoparticles critically impacts antigen binding and immune cell interactions via Fc receptors.
Current methods lack direct quantification of antibody orientation on nanoparticle surfaces.

Purpose of the Study:

To develop a generic, multiplexed methodology for simultaneous imaging of Fab and Fc exposure on antibody-conjugated nanoparticles.
To quantitatively assess antibody orientation and its heterogeneity using super-resolution microscopy.
To correlate nanoparticle characteristics and conjugation strategies with antibody domain exposure and biological function.

Main Methods:

Utilized super-resolution microscopy, specifically two-color DNA-PAINT imaging.
Employed Fab-specific Protein M and Fc-specific Protein G probes conjugated to single-stranded DNA.
Quantified antibody sites per particle and analyzed particle size effects on antibody coverage.

Main Results:

Presented a novel method for simultaneous, quantitative imaging of Fab and Fc exposure on nanoparticles.
Highlighted significant heterogeneity in antibody orientation across nanoparticle surfaces.
Demonstrated that conjugation strategies modulate Fab and Fc exposure, impacting antibody-dependent cell-mediated phagocytosis (ADCP).

Conclusions:

The developed super-resolution microscopy technique provides a universal tool for characterizing antibody-conjugated nanoparticles.
Quantitative data on antibody orientation improves understanding of structure-activity relationships in targeted nanomedicine.
This method enables tuning of nanoparticle conjugation for specific biomedical applications.