Click-Ready Lipids Explained: Maleimide, Azide/DBCO, and TCO/Tetrazine

Introduction 

Click-Ready lipids make it easy to attach ligands, dyes, and probes to nanoparticles (LNPs) and liposomes without complex chemical processes or harsh reaction conditions. Molecules conjugated through these chemistries exhibit high selectivity, compatibility to aqueous environments, and minimal by-product formation. In practice, the functional group “clicks” to a complementary handle on another molecule such as a ligand, dye, label, or tag. 

For drug delivery scientists, click-ready lipids streamline nanoparticle surface functionalization, enabling precise attachment of antibodies, peptides, or polymers for targeting, imaging, and controlled release. 

This overview highlights three of the most widely used “click-ready” pairs—maleimide/thiol, azide/DBCO, and TCO/tetrazine—that enable efficient, bio-orthogonal conjugation and how they can be applied for effective delivery with lipid-based systems. 

Click-ready lipids offer distinct advantages over traditional alternatives.  They enable site-selective coupling in aqueous solutions, allowing cargos to be attached after particle formation. Additionally, they help maintain small particle sizes with low polydispersity, unlike older methods which utilize bulky affinity pairs. 

Maleimide-Thiol

DSPE-PEG (2000) Maleimide reacts selectively with free thiol (-SH). A common example is the maleimide functional group clicking with a cysteine residue on a peptide. This chemistry is widely used because it is site selective, mild, and efficient at a neutral pH. Peptides can also be tagged with fluorescent attachments to facilitate tracking. 

Best practices for this chemistry include:

• Maintain pH between 6.5-7.0 in PBS or HEPES buffer
• Use a non-thiol reducer such as TCEP to minimize side reactions 

Azide-DBCO

DSPE-PEG (2000)-DBCO reacts rapidly and selectively with Azide-modi fied partners via copper-free strain-promoted azide–alkyne cycloaddition (SPAAC). A common use is clicking a Azide-tagged peptide, sugar, or fluorophore onto azide-bearing particles to add targeting or tracking without harsh conditions. This pair is favored because it’s bio-orthogonal, catalyst-free, and efficient near neutral pH, making it ideal for post-formulation labeling. 

Best practices: 

• Work in PBS/HEPES at pH ~6.8–7.4, 
• Keep organic co-solvent low (e.g., ≤10% DMSO) 
• Use a slight excess of the Azide reagent 
• Protect from prolonged light/heat
• Finish with SEC/desalting to remove unreacted label 

TCO-Tetrazine

DSPE-PEG (2000)-TCO reacts extremely rapidly with tetrazine-modified partners via the inverse-electron-demand Diels–Alder (IEDDA) reaction. This chemistry is ideal for post-formulation labeling at low concentrations or when very short reaction times are required under neutral, aqueous conditions. 

Best practices (TCO > Tetrazine):

• Work in PBS or HEPES at pH ~6.8–7.4 
• Use a slight excess of tetrazine
• Protect from light/heat to avoid potential oxidation 
• Purify by size exclusion or desalting to remove unreacted tetrazine
• Avoid prolonged exposure to high thiol or oxidant levels

This reaction can also proceed in reverse: DSPE-PEG (2000)-Tetrazine pairs with TCO-tagged peptides, sugars, or dyes using the same IEDDA click. This pairing is particularly useful when rapid, high-yield conjugation is needed without altering particle size or polydispersity.

Best practices (Tetrazine > TCO):

• Favor neutral buffers, keep organic co-solvent low (≤10% DMSO if needed)
• Use a slight excess of TCO
• Shield from light/oxygen
• Run at room temperature or cooler
• Finish with SEC/desalting.