Decoupling Physisorption from Chemisorption in Clickable Lipid Nanoparticles.

Area of Science:

• Bioconjugation Chemistry
• Nanoparticle Drug Delivery
• Surface Chemistry

Background:

• Antibody conjugation is critical for targeted lipid nanoparticle (LNP) delivery systems.
• Click chemistry is a common method for achieving antibody-LNP conjugation.
• Accurate measurement of covalent antibody-LNP bonds is essential for reliable targeting.

Purpose of the Study:

• To investigate artifacts in antibody-LNP conjugation measurements using click chemistry.
• To elucidate the role of hydrophobic interactions in antibody binding to LNPs.
• To establish reliable methods for quantifying covalent antibody-LNP conjugation.

Main Methods:

• Utilized click chemistry with dibenzocyclooctyne (DBCO) linkers for antibody conjugation to LNPs.
• Assessed antibody binding via chromatographic methods and plasma incubation stability.
• Compared conjugation efficiency on hydrophobic LNPs versus less hydrophobic liposomes.
• Evaluated the impact of substituting DBCO with bicyclononyne (BCN).

Main Results:

• Dibenzocyclooctyne (DBCO) linker's hydrophobicity drives significant nonspecific antibody physisorption onto LNPs.
• This physisorption leads to artificially high conjugation efficiencies measured by chromatography.
• Physisorbed antibodies are displaced by plasma, while covalently bound antibodies remain attached for effective in vivo targeting.
• Less hydrophobic liposomes show azide-dependent conjugation, and BCN reduces nonspecific binding.

Conclusions:

• Hydrophobicity is a critical, previously unrecognized variable in antibody-LNP conjugation.
• Current click chemistry methods can overestimate covalent antibody attachment to LNPs.
• New standards are needed for accurate, reproducible quantification of targeted LNPs.