Networked Cluster Formation via Trigonal Lipid Modules for Augmented Ex Vivo NK Cell Priming

Abstract

Current cytokine-based natural killer (NK) cell priming techniques have exhibited limitations such as the deactivation of biological signaling molecules and subsequent insufficient maturation of the cell population during mass cultivation processes. In this study, we developed an amphiphilic trigonal 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE) lipid-polyethylene glycol (PEG) material to assemble NK cell clusters via multiple hydrophobic lipid insertions into cellular membranes. Our lipid conjugate-mediated ex vivo NK cell priming sufficiently augmented the structural modulation of clusters, facilitated diffusional signal exchanges, and finally activated NK cell population with the clusters. Without any inhibition in diffusional signal exchanges and intrinsic proliferative efficacy of NK cells, effectively prime NK cell clusters produced increased interferon-gamma, especially in the early culture periods. In conclusion, the present study demonstrates that our novel lipid conjugates could serve as a promising alternative for future NK cell mass production.

Keywords: biomaterial-mediated clustering; ex vivo NK cell priming; hydrophobic insertion; lipid-polymer conjugate; membrane contact.

Figure 1

Schematic illustration of trigonal-linker (TL)-mediated NK cell clustering for ex vivo priming. (A) The synthesis route and complete structure of the TL module involved the conjugation of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG-NH₂) with trimesic acid (TMA), resulting in the formation of (DSPE-PEG-NH)₃-T. (B) The process of TL module-mediated NK cell clustering and priming. The upregulation of cytokine secretion, a crucial indicator of cell priming, was successfully achieved through the promotion of spheroid formation by (DSPE-PEG-NH)₃-T.

Figure 2

Fourier-transform infrared (FTIR) spectroscopic analysis of trimesic acid (TMA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG-NH₂), and synthesized trigonal-linker module, DSPE-PEG-NH branching with TMA ((DSPE-PEG-NH)₃-T).

Figure 3

Proton nuclear magnetic resonance (¹H-NMR) spectra of trimesic acid (TMA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG-NH₂), and DSPE-PEG-NH branching with TMA ((DSPE-PEG-NH)₃-T). The NMR spectrum results were recorded in dimethyl sulfoxide (DMSO)-d₆ solvent.

Figure 4

NK cell viability with the module (DSPE-PEG-NH)₃-T for 6 h. Cell viability was evaluated in NK cells with a 0~200 µg/mL concentration of the module. In the subsequent experiment, cells were treated with (DSPE-PEG-NH)₃-T at a 40 µg/mL concentration. The data were expressed as mean ± standard deviation (SD) based on a triplicate measurement. (ns: not significant).

Figure 5

NK spheroid volume and morphology analysis. NK spheroid formation image at 6 h in (A) the NK cell group and (B) the TLNK cell group. (C) Cell circularity with (DSPE-PEG-NH)₃-T for 6 h. (D) Volume quantification with (DSPE-PEG-NH)₃-T for 24 h and 48 h. A single spheroid was made up of 5 × 10³ NK cells. The area, perimeter, length, and width of spheroids were measured by ImageJ software 1.53e. Also, the spheroid circularity and volume were calculated with Formulas (2) and (3). The data were expressed as mean ± standard deviation (SD) based on a triplicate measurement. (* p < 0.05, ns: not significant).

Figure 6

IFN-γ secretion in TLNK cells after (A) 6 h and (B) 24 h of spheroid formation. All groups were treated with 1 µg/mL lipopolysaccharide (LPS) for 24 h for cytokine inducement. IFN-γ secretion was detected by ELISA and the data are expressed as mean ± standard deviation (SD) based on a triplicate measurement. (* p < 0.05).