Influence of multivalent nitrilotriacetic acid lipid-ligand affinity on the circulation half-life in mice of a liposome-attached His6-protein.

Metal chelation-ligand interactions, such as occur between nitrilotriacetic acid (NTA)-nickel and multihistidines, enable the noncovalent attachment of histidine-modified proteins to liposomes and other particles. We compared three lipids: a mono-NTA lipid (ca. 10 microM affinity) and two tris-NTA lipid derivatives (ca. 3 nM and 0.2 nM affinity) in their ability to retain two different his(6)-containing proteins on NTA-liposomes in the presence of serum or plasma and after intravenous injection in mice. At nanomolar affinities, the off-rate of a his(6)-ligand is sufficiently long so that his(6)-proteins attached to particle surfaces will remain with the particle for hours; thus, we hypothesized that the increased his(6) affinity of multivalent NTA-modified liposomes would retain his(6)-proteins longer both in vitro and in vivo. For each of the three lipids, we found a robust association and complete activity retention of two his(6)-modified proteins: a far red-fluorescent protein, monomeric Katushka (mKate), and a prodrug-converting enzyme, yeast cytosine deaminase (yCD). Proteins associated more tightly in vitro with tris-NTA liposomes than with mono-NTA liposomes in the presence of refiltered fetal calf serum and mouse plasma. Free yCD exchanged with previously associated mKate for tris-NTA binding sites on the liposome surface. This exchange was due to the exchange of the proteins for NTA occupancy and not due to the exchange of tris-NTA lipid out of the liposome. The amount of yCD on the surface was similar if the proteins were co-associated or if mKate was pre-associated. This exchange confirms that NTA associated proteins are in a dynamic state and can exchange with multihistidine proteins in the biological milieu. There was no difference in circulation time of the protein when it was intravenously administered by itself or attached to any of the NTA-modified liposomes because in vivo the protein was rapidly released from the NTA liposomes. Upon recovery from blood, liposomes containing tris-NTA accumulated a different plasma protein profile than control liposomes, suggesting that Ni-NTA specifically interacts with some plasma proteins. The reason for the rapid protein dissociation from the liposome in vivo is not clear; it could be due to displacement by endogenous histidine-containing proteins or to natural chelators that remove nickel from the NTA. Regardless of the cause, improvements in chelator or ligand design are needed before metal chelation will be capable of retaining histidine-modified proteins on NTA liposomes after in vivo administration.