pH-degradable imidazoquinoline-ligated nanogels for lymph node-focused immune activation

Abstract

Agonists of Toll-like receptors (TLRs) are potent activators of the innate immune system and hold promise as vaccine adjuvant and for anticancer immunotherapy. Unfortunately, in soluble form they readily enter systemic circulation and cause systemic inflammatory toxicity. Here we demonstrate that by covalent ligation of a small-molecule imidazoquinoline-based TLR7/8 agonist to 50-nm-sized degradable polymeric nanogels the potency of the agonist to activate TLR7/8 in in vitro cultured dendritic cells is largely retained. Importantly, imidazoquinoline-ligated nanogels focused the in vivo immune activation on the draining lymph nodes while dramatically reducing systemic inflammation. Mechanistic studies revealed a prevalent passive diffusion of the nanogels to the draining lymph node. Moreover, immunization studies in mice have shown that relative to soluble TLR7/8 agonist, imidazoquinoline-ligated nanogels induce superior antibody and T-cell responses against a tuberculosis antigen. This approach opens possibilities to enhance the therapeutic benefit of small-molecule TLR agonist for a variety of applications.

Keywords: Toll-like receptor; dendritic cells; lymph node; nanotechnology; vaccine.

Fig. 1.

Assembly of degradable immune-stimulatory nanogels. (A) Schematic overview and (B) corresponding chemical structures of nanogel assembly. (a) Block copolymers self-assemble in DMSO into nanoparticles. (b) Covalent ligation of TLR7/8 agonist (green) and cross-linking. (c) Conversion of residual pentafluorophenyl ester with 2-ethanolamine yielding fully hydrated nanogels after transfer to the aqueous phase.

Fig. 2.

(A) Molecular weight distributions of poly(mTEGMA) (blue line), poly(mTEGMA-b-PFPMA) (red dotted line), and poly(mTEGMA-b-PFPMA) after removal of the dithiobenzoate end group (red line). (B) Size distribution curves (volume mean diameter) measured in DMSO of poly(mTEGMA-PFPMA) (red line), measured in PBS after cross-linking with 2,2-bis(aminoethoxy)propane and conversion of residual activated PFP esters with 2-aminoethanol (blue line), and measured in PBS after conversion of residual activated PFP esters with 2-aminoethanol, but without cross-linking (blue dotted line). (C) Evolution of light scattering count rate (C1) and Z-average hydrodynamic diameter (C2) in response to pH 5 (red line) and pH 7.4 (blue line), respectively, as function of time. (C3) Size distribution curves (volume mean diameter) of the nanogels at different time points during incubation at pH 5 (red line) and pH 7.4 (blue line), respectively.

Fig. 3.

(A) Cellular association of TMR-labeled polymer [poly(mTEGMA-b-HEMAm)] and ketal-cross-linked poly(mTEGMA-b-HEMAm)-based nanogels by DCs at 4 °C (red) and 37 °C (blue), expressed as (A1) percentage of positive cells and (A2) mean cellular fluorescence intensity (MFI) (n = 3). (B) Confocal microscopy images of DCs pulsed at 37 °C with TMR-labeled (green channel) (B1) nanogels and (B2) (non-cross-linked) polymer. Cell membranes were labeled with AF488-cholera toxin B (red channel) and cell nuclei were labeled with Hoechst 33258 (blue channel). Panels (a) depict a confocal xy section with the corresponding orthogonal xz and yz planes. Panels (b) depict maximum intensity projections (MIP) of the full recorded Z-stack. The images were recorded using identical settings for excitation power, detection sensitivity and contrast. (Scale bars, 10 µm.) (C) FACS analysis of the induction of DC maturation by soluble IMDQ (blue), IMDQ-ligated polymer (green), and IMDQ-ligated nanogels (red) for different concentrations of IMDQ (filled bars). The corresponding non–IMDQ-ligated controls are depicted by the hollow bars and correspond to the same concentration of DMSO to solubilize IMDQ in PBS and the same concentration of polymer, respectively, nanogels as in the IMDQ-ligated samples. Data are expressed as (C1) the percentage of MHCII^hi and CD80^hi cells and the mean MFI of the DCs in the (C2) MHCII and (C3) CD80 gate. n = 3.

Fig. 4.

(A) In vivo luminescence in IFN-β reporter mice. (A1) Images recorded at 4, 8, and 24 h following injection of soluble IMDQ and nanogel-ligated IMDQ in the footpad (each at 10-µg IMDQ equivalents). (A2) Quantification of (A2, a) the total luminescence in the dashed rectangular region of interest (ROI) and (A2, b) the ratio of the luminescence in the draining popliteal lymph node (DLN) and or injection site in the foot pad (circular ROIs) versus the total luminescence (rectangular ROI) (n = 3). (B) FACS analysis, 24 h postinjection, of the DLN, depicting (B1) the cell numbers and (B2) percentage/MFI of nanogel-positive cells. (n = 3). (C1) Confocal images, 24 h postinjection, of the DLN of WT vs. CCR7^−/− mice injected with IMDQ nanogels and blank nanogels (each at 100-µg nanogel or 10-µg IMDQ equivalents, respectively). Note that images corresponding to IMDQ-nanogels were stitched due to the large dimension of the DLN by IMDQ activation. For clarity the green (i.e., nanogel) channel is depicted next to the multicolor image. (Scale bars, 100 µm.) (C2) FACS analysis, 24 h postinjection, of the DLN of WT vs. CCR7^−/− mice for the presence of IMDQ nanogels and nanogels. (D) Levels of PPE44-specific IFN-γ secreting CD8 (D1) and CD4 (D2) T cells in the spleen measured by ELISPOT in immunized WT mice (n = 4). (E1 and E2) Antibody titers against PPE44 measured by ELISA in the serum of immunized WT mice (n = 5).