Clickable Methyltetrazine-Indocarbocyanine Lipids: A Multicolor Tool Kit for Efficient Modifications of Cell Membranes

Cell-based therapeutics are one of
the most promising and exciting
breakthroughs in modern medicine. Modification of the cell surface
with ligands, biologics, drugs, and nanoparticles can further enhance
the functionality. Previously, we described the synthesis of a dioctadecyl
indocarbocyanine Cy3 analog (aminomethyl-DiI) for efficient and stable
modification (painting) of mouse erythrocytes with small molecules,
enzymes, and biologics. Here, we synthesized a near-infrared aminomethyl
dioctadecyl derivative of Cy7 (aminomethyl-DOCy7) and systematically
compared it to aminomethyl-DiI as an anchor for the modification of
human erythrocytes, Jurkat cells, and primary T cells with immunoglobulin
G. To enable copper-free click chemistry modification of cell membranes,
we conjugated a methyltetrazine (MTz) group to the amino-indocyanine
lipids via a polyethylene glycol (PEG) linker. DOCy7–PEG3400–MTz
showed over 99% modification efficiency of human red blood cells (RBCs)
at 25 μM. Reaction of trans-cyclooctene (TCO)
modified immunoglobulin G (IgG) with DOCy7–PEG4–MTz-modified
RBCs (2-step method) resulted in ∼80,000 IgG molecules per
erythrocyte, whereas modification with a preconjugated DOCy7–PEG3400–IgG
construct (1-step method) resulted in ∼20,000 IgG molecules
per erythrocyte as detected by immuno dot-blot. The number of IgG/RBC
was controlled by the concentration of IgG. The incubation of RBCs
with DiI–PEG3400–MTz resulted in a similar number of
IgG/RBC. Modification of the T-lymphocyte cell line Jurkat with IgG
resulted in ∼1 × 106 IgG/cell with the 1-step
and 2-step methods, and the efficiency was similar for DOCy7 and DiI
constructs. Finally, we used DOCy7 and DiI constructs to demonstrate
efficient modification of primary CD3+T cells from healthy donors.
In conclusion, click indocarbocyanine conjugates represent a novel
multicolor chemical biology tool kit for efficient surface modification
of different cells types and can be used for potential imaging and
drug delivery applications involving engineered cells.