Influence of Multivalent Nitrilotriacetic Acid Lipid−Ligand Affinity on the Circulation Half-Life in Mice of a Liposome-Attached His₆-Protein

Abstract

Metal chelation-ligand interactions, such as occur between nitrilotriacetic acid (NTA)-nickel and multihistidines, enable the non-covalent attachment of histidine-modified proteins to liposomes and other particles. We compared three lipids: a mono-NTA lipid (circa 10 uM affinity) and two tris-NTA lipid derivatives (circa 3 nM and 0.2 nM affinity) in their ability to retain two different his6-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 his6-ligand is sufficiently long so that his6-proteins attached to particle surfaces will remain with the particle for hours; thus, we hypothesized that the increased his6 affinity of multivalent NTA-modified liposomes would retain his6-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 his6-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.

Antibody Response to Polyhistidine-Tagged Peptide and Protein Antigens Attached to Liposomes via Lipid-Linked Nitrilotriacetic Acid in Mice

“…Although metal chelation via NTA-Ni(II)-His has been suggested as a site-specific, nondestructive approach to particulate delivery of polyhistidine-tagged antigens (9,35), concerns that the micromolar affinity of monovalent NTA for hexahistidine may be too weak for in vivo applications have arisen (22,27,37,45). We hypothesized that the increased affinity of trivalent NTA for polyhistidine (K D , ϳ1 nM) would translate to increased liposome association and enhanced antibody titers compared to a monovalent NTA linkage (K D , ϳ10 M).…”

“…This would result in dissociation of the histidine-tagged antigen from the liposome. Alternatively, the His-tagged antigen may be exchanged with other polyhistidine-containing proteins in vivo; we recently observed that His-tagged proteins attached to liposomes via tris-NTA lipids are readily exchanged with other His-tagged proteins in the bulk solution (37). Thus, although the fate of the NTA-Ni(II)-His interaction in vivo is unclear, increased affinity up to the low nanomolar range does not translate to greater antibody responses in our system.…”

“…Potential modifications include further increasing NTA valency (27), modifying the flexibility and length of the spacer between the mono-NTA and the scaffold (21), and incorporating hydrophobic shielding groups that impede removal of the chelated metal. These approaches are discussed in greater detail in recent publications from our laboratory (21,37). We are also investigating branched and multivalent polyhistidine architectures as novel adaptors for NTA-Ni(II) (unpublished results).…”

“…This approach has shown promise for in vivo delivery; most notably, plasma membrane vesicles to which polyhistidinylated dendritic cell-targeting moieties and costimulatory molecules were engrafted via trivalent NTA elicited functional antitumor immunity upon administration to mice (44). However, the retention of His-tagged proteins in trivalent NTA-containing formulations in vivo remains unclear; we recently found that despite stable association of His-tagged proteins with tris-NTA-containing liposomes in serum in vitro, the proteins dissociate within minutes upon intravenous administration (37). Thus, more work is needed to clarify the relationship between NTA-Ni(II)-His affinity in vitro and biological activity in vivo.…”

Multivalent and Flexible PEG-Nitrilotriacetic Acid Derivatives for Non-covalent Protein Pegylation

“…As demonstrated in previous studies, mono-NTA derivatives and (His6)-tagged proteins have a weak interaction-the K D is about 10 μM-but it can be significantly increased by exploiting the multivalency of derivatives bearing several molecules of NTA (16)(17)(18). Szoka and colleagues recently proposed a new tris-NTA derivative, based on a dendritic lysine scaffold, that fits perfectly with the structure of His6 tag and yields a K D of about 20 nM (18)(19)(20). This approach is suitable for His6-tagged proteins, and it has been successfully used for the preparation of surface plasmon resonance (SPR) chips with high NTA density (21).…”