Multifunctional nanorods serving as nanobridges to modulate T cell-mediated immunity

Abstract

Electrodeposited nanorods serving as multivalent bridges were fabricated and surface-decorated with ligands for immune cells. Gold and nickel solutions were sequentially electrodeposited on nanoporous anodized disc templates and the template was dissolved to retrieve bisegmented nanorods with different lengths. Gold and nickel segmented nanorods were surface-immobilized with mannose and RGD peptides to prepare immune-cell recruiting nanorods. Surface-functionalization of nanorods were confirmed by fluorescence-labeling of each ligands and confocal microscopy. Dendritic cells and T cells were co-incubated with the surface-functionalized nanorods, and the proximity between the nanorods and the immune cells was visualized by variable pressure scanning electron microscopy and confocal microscopy. The long nanorods were associated with the immune cells, whereas the shorter nanorods were rather endocytosed by cells, suggesting a feasibility of the longer nanorods as bridging for the cells. Cytokine releases from the immune cells were monitored by cultivating lipopolysaccharide-activated dendritic cells with T cells. Interleukine-2 and interferon-γ release profiles showed a strong correlation with the length of the nanorod, where the 4 μm nanorods induced the highest levels of cytokine release compared to 1 or 2 μm nanorods. Thus, we concluded that the proximity of the immune cells increased by bridging the immune cells with the nanobridging system, which subsequently increased cytokine release by facilitating the antigen presentation process.

Figure 1

Preparation of Au/Ni nanorod for selective immobilization of mannose and RGD peptide (Man/RGD NR) and description of ‘nano-bridge’ for enhanced T cell-mediated immune responses. (A) Functionalization of mannose and RGD peptides for surface-immobilization. After pegylation of mannose, the terminal amine group of Man-PEG-NH2 was thiolated with 2-imminothiolane to prepare Man-PEG-SH. RGD-PEG-COOH was synthesized by conjugating hetero-functional PEG (NH₂-PEG-COOH) to the activated GRGDS peptides. (B) Selective immobilization of Au and Ni segments with the functionalized mannose and RGD peptides. After sequential electrodeposition of Au and Ni, Man-PEG-SH and RGD-PEG-COOH were added for selective modification by Au-thiol and Ni-carboxylic acid reactions. (C) BMDC and Jurkat cell-recognizable ‘nano-bridge’ for facilitating antigen presentation. By bridging between BMDC and Jurkat cell, the antigen presentation is facilitated and T cell mediated immune response is fortified.

Figure 2

Visualization of Mannose/RGD moieties-immobilized nanorods (Man/RGD NR) with different lengths (NR 1 = 1μm, NR 2 = 2μm, and NR 4 = 4μm). Back-scattered SEM images (A) shows bi-segmented nanorods composed of Au (white) and Ni segment (dark grey) of fluorescently-labeled Man/RGD NR. In CLSM images (B), where Alexa 647-labeled Mannose and FITC-labeled RGD moieties are shown in red and green, respectively. The illustrations of the Man/RGD NR are also represented (C). The scale bars of all microscopic images are 2μm.

Figure 3

Visualization of the immune cells bridged with Man/RGD NRs. The proximity of the nanorods was confirmed both by VP-SEM (A, E and G) equipped with EDS (B, E, and H) and CLSM (C, F, and I). Man/RGD-NRs were incubated with T cells for 3h and LPS-activated BMDCs were then added. After 18h, Man/RGD-NR associated with BMDC and T cell was visualized by VP-SEM on a cool-stage at −14°C (left). While the inset images are in low magnification, the magnified images of the white circles in the inset were also shown. EDS spectrum of the indicated spots (□/+) in SEM images (middle). The peaks of Ni (0.9 keV) and Au (2.3 keV) were assigned to determine the orientation of the Man or RGD. In CLSM images, BMDC (yellow) and T cell (blue) were shown with Man/RGD-NR (white arrow) with RGD (green) and mannose (red) (right). Orthogonal views are also presented on the upper and the right to highlight the location of the nanorods. All scale bars in the images indicate 2μm.

Figure 4

Cytokine release from T cell upon administration of nanorods. Jurkat cells were incubated with Man/RGD NRs at the presence of CD11c⁺ BMDCs in serum free media for 24h. Man/RGD NRs were pre-incubated with Jurkat cells for 3h and CD11c⁺ BMDCs were added (number ratio of NR: Jurkat cell: BMDC=50:5:1). (A) Interleukine-2 (IL-2) and (B) interferon-γ (IFN-γ) levels of the released medium were determined by ELISA as described in Methods. In (C) and (D), RGD receptor (α_vβ₃) on T cells or mannose receptors on BMDC were pre-blocked in the cell culture medium containing 0.2 mM RGD peptides or 1.2 mM mannose to disturb the association of Man/RGD NR 4 toward immune cells. (C) IL-2 and (D) IFN-γ levels were measured with the same method. * indicates statistical significance (p<0.05).