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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.
ACS Nano
|June 7, 2023
Summary
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.
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.
