Glyco-Nanoadjuvants: Impact of Linker Length for Conjugating aSynthetic Small-Molecule TLR7 Ligand to Glyco-Nanoparticles onImmunostimulatory Effects br

Immunotherapy has become a powerful clinicalstrategy for treating infectious diseases and cancer. Synthetic small-molecule toll-like receptor 7 (TLR7) ligands are attractivecandidates as immunostimulatory agents for immunotherapy.TLR7 is mainly localized in intracellular endosomal compartmentsso that the formulation of their small-molecule ligands withmacromolecules enhances endocytic uptake of TLR7 ligands andimproves the pharmaceutical properties. Previously, we demon-strated that gold nanoparticles co-immobilized with a TLR7 ligandderivative, that is, a conjugate of synthetic small-molecule TLR7ligand (1V209) and thioctic acid (TA) via 4,7,10-trioxa-1,13-tridecanediamine, and alpha-mannose (1V209-alpha Man-GNPs: glyco-nanoadjuvants) significantly enhances immunostimulatory effects. Inthe present study, we designed a second-generation glyco-nanoadjuvant that possesses a poly(ethylene glycol) (PEG) chain as aspacer between 1V209 and GNPs and investigated the impact of linker length in 1V209 derivatives on the immunostimulatoryactivities. We used different chain lengths of PEG (n= 3, 5, 11, or 23) as spacers between 1V209 and thioctic acid to prepare four1V209-alpha Man-GNPs. In thein vitrostudy using primary mouse bone-marrow-derived dendritic cells, 1V209-alpha Man-GNPs thatimmobilized with longer 1V209 derivatives, especially the 1V209 derivative possessing PEG23 (1V209-PEG23-TA), showed thehighest potency toward induction both for interleukin-6 and type I interferon production than those derivatives with shorter PEGchains. Furthermore, 1V209-alpha Man-GNPs that immobilized with 1V209-PEG23-TA showed significantly higher adjuvant effects forinducing both humoral and cell-mediated immune responses against ovalbumin in thein vivoimmunization study. These resultsindicate that the linker length for immobilizing small-molecule TLR7 ligand on the GNPs significantly affects the adjuvant activity of1V209-alpha Man-GNPs and that 1V209-alpha Man-GNPs immobilized with 1V209-PEG-23-TA could be superior adjuvants for immunotherapies.