Table 1.

Summary of Covalent Reactions for Cell Surface Engineering

Cell Surface Functional Group	Cross-linker	Reaction Conditions	Benefits/Limitations	Ref.
Native Amines		Incubation with NHS ester in pH 8.0 for 20 min – 1 hr at room temperature to 37 °C	Most Common conjugation method Stable amide bond formation Slower reaction than cyanuric chloride	44, 131, 133, 136, 138, 144, 145, 183, 207
		Incubation with benzotriazole carbonate in pH 8.0 for 1 hr at room temperature	Stable amide bond formation Slower reaction than cyanuric chloride More reagent required	145
		Incubation with 2-iminothiolane and malimidophenyl in pH 7.4 for 2 hr at room temperature	Iminothiolane can reach buried amine groups Provides a 8 Å extension away from surface making attachment of bulky groups possible Will not alter net surface change of functionalized surface	146
		Incubation with cyanuric chloride in pH 8.7 for 20–30 min	Highly reactive, shorter reaction times Introduction of bulky triazine group Possible over functionalization	143 – 5
Native Thiols		Incubation with 2-pyridyl-dithio group for 4 hr at 37 °C	Close to quantitative conjugation High cell viability(~ 85%) Limited to the number of exofacial protein thiols (cell dependent) Possible internalization of conjugated group	78
		Incubation with malimidophenyl group for 30 min at 37 °C	Cellular function maintained Limited to the number of exofacial protein thiols (cell dependent) Possible internalization of conjugated group	185
Glycans		Incubation with NaIO4 for 15 min at 4 °C followed by Incubation with hydrazine linker for 30 min	Limited cytotoxicity ≤ 20% Maintained cell proliferation High incorporation of ligand	197–9