Targeted Delivery of Adamantylated Peptidoglycan Immunomodulators in Lipid Nanocarriers: NMR Shows That Cargo Fragments Are Available on the Surface

Abstract

We present an in-depth investigation of the membrane interactions of peptidoglycan (PGN)-based immune adjuvants designed for lipid-based delivery systems using NMR spectroscopy. The derivatives contain a cargo peptidoglycan (PGN) dipeptide fragment and an adamantyl group, which serves as an anchor to the lipid bilayer. Furthermore, derivatives with a mannose group that can actively target cell surface receptors on immune cells are also studied. We showed that the targeting mannose group and the cargo PGN fragment are both available on the lipid bilayer surface, thereby enabling interactions with cognate receptors. We found that the nonmannosylated compounds are incorporated stronger into the lipid assemblies than the mannosylated ones, but the latter compounds penetrate deeper in the bilayer. This might be explained by stronger electrostatic interactions available for zwitterionic nonmannosylated derivatives as opposed to the compounds in which the charged N-terminus is capped by mannose groups. The higher incorporation efficiency of the nonmannosylated compounds correlated with a larger relative enhancement in immune stimulation activities upon lipid incorporation compared to that of the derivatives with the mannose group. The chirality of the adamantyl group also influenced the incorporation efficiency, which in turn correlated with membrane-associated conformations that affect possible intermolecular interactions with lipid molecules. These findings will help in improving the development of PGN-based immune adjuvants suitable for delivery in lipid nanoparticles.

Structural Fine-Tuning of Desmuramylpeptide NOD2 Agonists Defines Their In Vivo Adjuvant Activity

“…For example, addition of lipophilic adamantane groups to peptidoglycan fragments was shown to assist in the anchoring of these derivatives into the liposomal lipid bilayer, using NMR spectroscopy. 119 Likewise, straight-chain lipophilic anchors can be used for the same purpose, due to the extensive network of van der Waals interactions that they can form in the lipophilic bilayer of liposomes. Surprisingly, a reduction in chain length from C 18 to C 16 was shown to significantly increase the propensity for the undesired escape of lipids from liposomal membranes.…”

Design, Synthesis, and Biological Evaluation of Desmuramyl Dipeptides Modified by Adamantyl-1,2,3-triazole

“…Therefore, within this study, we have explored the modification of the D-Glu at the α-COOH group. In addition to better ligand binding to NOD2, the proposed design will also enable greater exposure of the adamantane structure for possible membrane anchoring, compared to those of ManAdDMP analogs [25,26].…”

Adamantane Containing Peptidoglycan Fragments Enhance RANTES and IL-6 Production in Lipopolysaccharide-Induced Macrophages

“…The more pronounced enhancement of pro-inflammatory cytokine and chemokine expression by l-AdTp compared to Man-l-AdTp could be explained by the negative influence of the hydrophilic mannose group on the incorporation efficiency into phospholipid vesicles. NMR studies of the PGN derivatives in model lipid assemblies have indeed confirmed that the non-mannosylated compounds have larger lipid incorporation efficiencies and penetrate the bilayer deeper than compounds with mannose groups [25].…”

“…Previous research on similar model PGN compounds with adamantyl-2-yl attachment incorporated into liposomes using atomic force microscopy revealed that the adamantane group is anchored into the lipid bilayer and that mannose residues are distributed at the surface of nanoparticles [26]. The localization and orientation of previously described adamantylated PGN derivatives in model lipid assemblies were also explored in detail using NMR spectroscopy, which showed the localization of bulky adamantane group in the interior of the bilayer and that of the D-iGln containing PGN fragment, as well as the presence of the mannose group on the surface of the bilayer [25].…”

“…Mannosylated liposomes preferentially target macrophages and dendritic cells, both in vitro and in vivo, and improve liposome internalization by macrophages and, consequently, enhance the immune response [25]. Hence, mannosylated nanoparticles formed by Man-l-AdTp could potentially be used for specifically targeting surface attached CLRs such as the MR. Receptor recognition of the mannosylated adamantylated PGN compounds, however, merits further investigation.…”